CN113180896A - Degradable support for preventing and treating esophageal stenosis - Google Patents
Degradable support for preventing and treating esophageal stenosis Download PDFInfo
- Publication number
- CN113180896A CN113180896A CN202110416365.2A CN202110416365A CN113180896A CN 113180896 A CN113180896 A CN 113180896A CN 202110416365 A CN202110416365 A CN 202110416365A CN 113180896 A CN113180896 A CN 113180896A
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- esophageal
- stenosis
- stent
- degradable
- degradable stent
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
- A61F2/02—Prostheses implantable into the body
- A61F2/04—Hollow or tubular parts of organs, e.g. bladders, tracheae, bronchi or bile ducts
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
- A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
- A61F2/02—Prostheses implantable into the body
- A61F2/04—Hollow or tubular parts of organs, e.g. bladders, tracheae, bronchi or bile ducts
- A61F2002/044—Oesophagi or esophagi or gullets
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
- A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
- A61F2/82—Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
- A61F2002/821—Ostial stents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
- A61F2210/00—Particular material properties of prostheses classified in groups A61F2/00 - A61F2/26 or A61F2/82 or A61F9/00 or A61F11/00 or subgroups thereof
- A61F2210/0004—Particular material properties of prostheses classified in groups A61F2/00 - A61F2/26 or A61F2/82 or A61F9/00 or A61F11/00 or subgroups thereof bioabsorbable
Abstract
The invention relates to a degradable stent for preventing and treating esophageal stenosis, belonging to the technical field of medical instruments. Comprises a film pasting material and a degradable bracket of genipin cross-linked chitosan collagen loaded with PLGA/triamcinolone acetonide sustained release microspheres; the periphery of the degradable stent is provided with a genipin cross-linked chitosan collagen film material loaded with PLGA and triamcinolone acetonide sustained release microspheres. The degradable stent is mainly used for preventing and treating benign stricture of the esophagus, in particular to a series of benign stricture problems of postoperative wound surfaces of esophageal precancer treated by Endoscopic Submucosal Dissection (ESD), postoperative anastomotic stricture after esophageal postoperative surgery, scar contracture stricture after esophageal chemical injury and the like.
Description
Technical Field
The invention relates to a degradable stent for preventing and treating esophageal stenosis, belonging to the technical field of medical instruments.
Background
Esophageal cancer is a high-grade digestive tract tumor in China, the statistics of cancer data in 2015 show that the number of new esophageal cancer cases in China is more than half of the total new cases in the world, and the esophageal cancer cases are one of the top five lethal tumors in China [1 ]. In recent years, the popularization of NBI (narrow band imaging) amplified endoscopic technology, ESD (endoscopic submucosal dissection) technology and early cancer screening of the digestive tract are benefited, so that the early diagnosis and treatment of esophageal cancer in China are greatly improved. ESD is a technology for endoscopic alimentary tract early cancer excision, has the characteristics of safety, minimally invasive property and the like on the basis of ensuring clean incisional margin, and researches show that ESD has similar survival benefit compared with surgical operation for esophageal mucosal cancer [2 ]. Benign stricture after esophageal lesion endoscopy operation, which is brought along with the popularization of ESD, is increased year by year. Due to the lumen structure of the esophagus, for patients with extensive mucosal dissection, the physiological scar repair process often causes irreversible luminal stenosis and further causes serious eating difficulty, and different researches show that the probability of significant stenosis of ESD patients with half-cycle or more of the esophagus under the condition of no intervention is about 70-90%, and the stenosis degree is positively correlated with the dissection radius range [3-4 ]. Although the cases are not malignant diseases, the patients basically lose labor capacity due to the fact that the patients cannot eat or even enter water, family care and even venous nutrition maintenance are needed, meanwhile, the survival expectation of the patients is far greater than that of the patients with the malignant diseases, so that more social medical resources are consumed in the course of the disease, and some patients finally have to perform gastrostomy, and the life of the patients is prolonged. If the esophageal stenosis after the ESD operation can be properly solved, the part of people can regain the health without damage, so that the health is completely integrated into the social life of normal people.
1. Current status of treatment of esophageal ESD postoperative strictures;
the molecular mechanism of restoration and healing of the wound surface after esophageal ESD operation and scar formation is complex, inflammation plays an important role in the repair and healing of the wound surface and scar formation, inflammatory cells are activated after tissues are subjected to various chemical and mechanical injuries, the healing and the repair of the wound surface are promoted, and when the process is short and ordered, the tissue reconstruction is complete; once the injury becomes persistent, the over-activated inflammatory cells, especially mononuclear macrophages secrete a large amount of inflammatory cytokines and growth factors (TNF-alpha, IL-6, TGF-beta 1) to act on fibroblasts to differentiate the fibroblasts into myofibroblasts, the differentiated myofibroblasts express a large amount of skeletal protein alpha-SMA, have the phenotype of partial myocytes to cause wound surface contraction, secrete a large amount of various types of collagen, and excessively deposited collagen forms collagen fibers to form fibrotic scars [5-6 ]. The esophagus becomes gradually narrower, about 2 weeks of stenosis is formed, and the shape is shaped by radial supporting force provided by the structure of the esophageal endotracheal cartilage, so that the wound surface is narrowed to a gap of only 1-2mm after the scar is formed [7 ]. The current treatment of esophageal ESD postoperative stenosis mainly comprises methods at the physical and mechanical level: under-endoscope dilation (such as balloon dilation and bougie dilation), radial dissection, and stent placement; and direct acting scar generation on the molecular mechanism level: the narrow section is locally injected with drugs such as triamcinolone acetonide and the like, or directly takes oral hormone to act systemically. Among them, endoscopic dilatation (such as saccule and bougie) requires multiple dilations in a short time to be effective, and has limited curative effect on complicated stenosis and high recurrence rate of stenosis [8 ]. The main mechanism of action of endoscopic radial incision in a narrow section is similar to endoscopic balloon or bougie dilatation, so that the risk is relatively high, and the current effect is not superior to endoscopic balloon dilatation and other treatments [9 ]. The metal stent is mainly implanted into a metal stent in clinic, which is a 'wang' method for treating complicated esophageal stenosis, but the metal stent is kept in a body as a foreign body for a long time, so that esophageal wall inflammation, mucosal hyperproliferation and esophagus injury are easily caused, restenosis is stimulated to occur, and dysphagia of a patient is caused. Therefore, in the case of stenosis after ESD operation, after the metal stent assists the esophagus to complete the remodeling, the stent has no significance of continuing to exist, and the stent needs to be recycled by secondary operation. However, after the stent is placed, the stent is easy to be embedded in the hyperplasia part due to the hyperplasia of epithelial cells and is often difficult to take out; meanwhile, the metal stent can cause secondary damage to the esophagus in the recovery process, other complications are caused, and the restenosis rate after the metal stent is placed for the first time is higher than 50% although being lower than that of an endoscopic dilation method [10 ]. One hotspot of improving the stent at the present stage is to design and develop a novel degradable esophageal stent, which supports stenosis in a lumen in a short term, and by controlling the degradation rate of materials, after the treatment mission of the stent is completed in the specific pathophysiological process of a human body, the stent disintegrates because of continuous degradation and loss of mechanical properties, finally degrades and disappears in vivo or is fragmented in gastrointestinal tract to be discharged, thereby avoiding complications caused by long-term foreign body influence and pain of taking out the stent [11 ]. The medicine has certain effect on systemic oral hormone and wound injection [12-13 ]. Its anti-fibrotic action derives from the inhibition of the function of inflammatory cells such as macrophages on the one hand and from the direct inhibition of fibroblast differentiation on the other hand [15 ]. Compared with the prior art, the ESD wound surface local injection of the long-acting hormone (triamcinolone acetonide) is simple and convenient to operate, the dosage of the drug is less, obvious systemic immunosuppression cannot be caused, and osteoporosis, femoral head necrosis, glycometabolism abnormality and the like are generated [16 ]. It follows that the most promising of the current approaches to the prevention of post-operative strictures in the esophagus ESD is the development of novel stents and precise topical application of long-acting hormones.
2. Domestic and foreign research status of degradable esophagus stent
Polylactic acid and polydioxanone are the most extensive materials with better biocompatibility and mechanical property in the research of the esophageal degradable stent. The degradation rate depends on the molecular weight, hydrophilicity and the pH value of the surrounding environment [17 ]. Foreign Saito Y et al have already studied the cases of implanting degradable stents after esophageal ESD surgery, but the sample size is a little. Similar to the common stent, the displacement rate of the degradable stent reaches 80 percent after one week, and the restenosis rate is about 50 percent. Although the research on the domestic degradable stent is followed, no product enters the clinic. It is not clear how long the degradable stent can be placed to achieve the desired effect, and it is generally assumed that at least 2-3 weeks are required for the degradable stent to be kept in place. The degradable stent has poor mechanical strength, so that the placement process is different from that of a metal stent and is complex, and the degradable stent needs to be fixed on the esophageal wall by an auxiliary means after being placed so as to prevent the degradable stent from shifting and falling off. A large sample size has yet to be investigated as a treatment for benign strictures in the esophagus [18-19 ]. Therefore, although the esophagus degradable stent overcomes the defects of foreign body inflammatory reaction, secondary extraction and the like of the traditional stent, the effect is not clear after being applied alone for a long time, and the mechanical property and the anti-displacement property of the esophagus degradable stent still need to be further improved, and the in-situ time is more than 2 weeks.
3. Research status of triamcinolone acetonide for preventing and treating esophageal ESD postoperative stenosis
Triamcinolone acetonide is the most commonly used hormone medicament for preventing and treating postoperative stenosis of ESD clinically at present, has strong and durable anti-inflammatory effect, and can inhibit mitosis of cells and synthesis of DNA (deoxyribonucleic acid), thereby inhibiting proliferation of fiber cells in scar tissues, reducing synthesis of collagen, increasing activity of collagenase, accelerating degradation of collagen and preventing adhesion and scar formation [20 ]. However, at present, the clinical application is mostly one-time local administration, the duration of the drug effect is short, and repeated administration is needed to possibly maintain the effective drug concentration for a long time. Although triamcinolone acetonide injected once in the operation can initially effectively reduce local inflammation and inhibit fibrous tissue proliferation, when the scar formation reaches a peak, the drug effect of the triamcinolone acetonide is weakened and does not reach an effective concentration, so that fibrosis and scar formation cannot be dried. If repeated injections are adopted, high concentration of local tissue drugs is easily caused, local muscular atrophy, telangiectasis, ulcer formation and the like are caused, and then delayed perforation risks exist. Furthermore, as fibrosis occurs at different stages, the doses required for local injection therapy with triamcinolone acetonide are in fact different, with the longer the scarring time the larger the drug dose required [21 ]. In view of this, in order to maintain a stable and effective drug concentration and ensure a sustained inflammation-inhibiting effect at each stage of stenosis formation, it is necessary to develop a long-acting sustained-release system of triamcinolone acetonide for preventing and treating postoperative stenosis in ESD.
4. Research situation of local precise sustained-release triamcinolone acetonide system
Polylactic-co-glycolic acid (PLGA) is a high molecular degradable organic compound polymerized from two monomers, has good biocompatibility, balling and film forming properties, is widely applied to wrapping of drugs and polypeptides, and is a drug adjuvant certified by FDA. In the uveitis model, it is reported that the PLGA/triamcinolone acetonide nano microsphere particles injected into the ball have longer release period and better treatment effect than naked drugs [22-23], so that a certain theoretical basis exists for constructing a slow release system by using PLGA as a coating material of the triamcinolone acetonide to treat esophageal stenosis. The chemical name of chitosan, polyglucosamine (1-4) -2-amino-B-D glucose, is widely used in the field of biomaterials because of its structure similar to that of natural extracellular matrix component mucopolysaccharide (GAG) [24] and its extremely high biocompatibility, degradability and safety [25 ]. It has a function of promoting the activation of inflammatory cells such as macrophages and polymorphonuclear leukocytes, and is considered to be a factor for promoting wound repair [26], and chitosan-based biomaterials have excellent functions in dermal defect repair and dermal burn repair [27-28 ]. Meanwhile, chitosan is proved to be a mucous membrane adhesion factor which can be spontaneously adhered to various animal cells [29], promote the permeation of macromolecular drugs in small intestine and nasal mucosa [30], and C.Padula et al also prove that the chitosan-rich microemulsion remarkably improves the permeation of triamcinolone acetonide in mucosa in a pig feed tract mucosa in vitro model [31 ]. In addition, although chitosan does not significantly inhibit fibrosis and scar formation, many composite materials have been used as an important component to prevent fibrosis formation due to its excellent biological properties, such as chitosan-hyaluronate hydrogel which significantly improves post-surgical adhesions [32 ]. Collagen (I, II, III, V, XI type) is the most important component of extracellular matrix, secreted by fibroblasts and myofibroblasts, and forms a reticular fiber structure to maintain the structural stability of tissues such as skin, tendon, submucosa, etc., so collagen has been used as an epidermal substitute [33], and a porous collagen scaffold loaded with autologous keratinocytes has been demonstrated to promote the revascularization and healing process of dermis, with excellent efficacy in the dermal wound repair process [34 ]. In the field of treating esophageal stenosis, application of collagen is reported, and Shigehisa Aoki et al prove that the re-epithelialization of a wound surface and the prevention of fibrotic stenosis can be effectively promoted by attaching an ESD wound surface through a high-density collagen pad subjected to transparentization treatment [35 ]. Therefore, the porous skeleton carrier prepared by utilizing the chitosan and the collagen is used for loading the PLGA microspheres wrapping the triamcinolone acetonide, so that the cell adhesion can be promoted theoretically, the drug permeation can be enhanced, and the formation of the fibrostenosis can be prevented. However, due to the special physicochemical properties of collagen, it often has no stable mechanical properties after molding and is easily degraded in a short period, so a specific cross-linking agent needs to be added to cross-link the chitosan and the free amino groups of collagen to obtain a stable structure. Genipin is a product of geniposide hydrolyzed by beta-glucosidase, and has certain crosslinking strength and low cytotoxicity as a crosslinking agent [36 ]. Meanwhile, genipin, as a natural anti-inflammatory factor, has been used in the treatment of cholestasis and hepatitis [37 ]. In addition, genipin also has the function of inhibiting fibrosis, and researches show that in the liver, genipin can effectively inhibit the activation of hepatic stellate cells so as to inhibit the formation of hepatic fibrosis [38], while in another in vitro model, genipin can obviously prevent the transformation of subconjunctival fibroblasts to myofibroblasts [39 ]. Therefore, genipin as a cross-linking agent of chitosan collagen not only can stabilize the material structure, but also is more likely to cooperate with hormone to play a role in anti-fibrosis and stenosis prevention. Therefore, the genipin cross-linked chitosan collagen film material loaded with PLGA/triamcinolone acetonide sustained release microspheres is researched and trial-produced, and is expected to have the property of inhibiting scar formation and not causing wound delayed healing.
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Disclosure of Invention
The invention aims to solve the technical problem of how to obtain a degradable stent for preventing and treating esophageal ESD postoperative stenosis. Provides a solid theoretical basis and practical experience for clinically and systematically treating the esophageal ESD postoperative stenosis, and solves the clinical urgent need of the disease population. The full tectorial membrane degradable support of medicine carrying that further degree of depth research and development fused medicine carrying slow release material and degradable support as an organic whole can promote the industrialization development of relevant degradable, medicine carrying material simultaneously.
In order to solve the problems, the technical scheme adopted by the invention is to provide a degradable stent for preventing and treating esophageal stenosis, which comprises a film pasting material of genipin cross-linked chitosan collagen loaded with PLGA/triamcinolone acetonide sustained release microspheres and the degradable stent; the periphery of the degradable stent is provided with a genipin cross-linked chitosan collagen film material loaded with PLGA/triamcinolone sustained release microspheres.
Preferably, the main body material of the degradable stent comprises polylactic acid, and the degradable stent is woven by an electrostatic spinning weaving process.
The invention provides application of a degradable stent for preventing and treating esophageal stenosis in treatment of benign esophageal stenosis.
The invention provides application of a degradable stent for preventing and treating esophageal stenosis in treatment of esophageal ESD postoperative stenosis.
The invention provides application of a degradable stent for preventing and treating esophageal stenosis in treatment of postoperative anastomotic stenosis of thoracic surgery and scar contracture stenosis after esophageal chemical injury.
Compared with the prior art, the invention has the following beneficial effects:
according to the degradable scaffold, the slow release of triamcinolone acetonide inhibits the proliferation of fibrocytes in scar tissues, reduces the synthesis of collagen, increases the activity of collagenase, accelerates the degradation of collagen, and prevents adhesion and scar formation; the product can be used for preventing and treating benign stricture of esophagus, especially postoperative wound surface of esophageal precancer treated by Endoscopic Submucosal Dissection (ESD), and a series of benign stricture problems such as postoperative anastomotic stricture after thoracic surgery and chemical injury of esophagus, scar contracture stricture after esophageal injury, etc. Provides a solid theoretical basis and practical experience for clinically and systematically treating the esophageal ESD postoperative stenosis, and solves the clinical urgent need of the disease population. The full tectorial membrane degradable scaffold with the medicine carrying has the advantages that the medicine carrying slow release material and the degradable scaffold are fused into a whole for further deep research and development, and meanwhile, the industrialized development of the related degradable and medicine carrying materials can be promoted.
Drawings
FIG. 1 shows a PLGA microsphere optical mirror structure (a, c and d) and an electron microscope structure (b, d) loaded with triamcinolone acetonide.
FIG. 2 shows an optical mirror structure (a) and an electron mirror structure (c and e) of a bare chitosan/collagen patch according to the present invention; the chitosan/collagen adhesive film loaded with triamcinolone acetonide/PLGA microspheres has a light mirror structure (figure b) and an electron microscope structure (figure d and figure f).
FIG. 3 is a graph showing the ability of a spherical patch to sustain sustained release of a drug in an in vivo environment; wherein a is a liquid chromatogram drug release curve chart of PLGA/triamcinolone acetonide drug-loaded microspheres; and b, a graph is a drug release curve chart of the chitosan/collagen patch loaded with the triamcinolone acetonide/PLGA microspheres.
FIG. 4 is a graph showing the effect of the patch material on inflammatory cell murine macrophages (Raw264.7) and fibroblast fibroblasts (L929); wherein a is the effect on macrophages (Raw264.7); panel b shows the effect on fibroblasts (L929).
FIG. 5 shows the WB assay of the effect of the patch material on inflammatory cells, murine macrophages (Raw264.7) and fibroblasts, fibroblasts (L929). Wherein a is the effect on macrophages (Raw264.7); panel b shows the effect on fibroblasts (L929).
FIG. 6 is a graph showing the effect of the patch material on the activity of murine macrophages (Raw264.7) which are inflammatory cells; there was no difference in macrophage activity between the 24h groups (panel a), but the C/C/P high group 48h had a promoting effect on macrophages (panel b), and the macrophages calcein markers were higher in both the C/C and C/P groups than in the control group (panel C), whereas both were lower in PI indices than in the control group (panel d): p is less than 0.05.
FIG. 7 shows the effect of the patch material on the activity of fibroblasts (L929). Wherein the C/C/P high group has fibroblast inhibiting effect 24h (figure a), the C/C/P high group has inhibiting effect except C/C low group compared with the control group in 48h, and the inhibiting effect of the C/C/P high group is stronger than that of the C/C/P low group (figure b); the calcein markers of the fibroblasts of group C/C and group C/P were lower than those of the control group (panel C), whereas both of the PI markers were higher than those of the control group (panel d): p is less than 0.05.
FIG. 8 is a graph showing the effect of a patch material on inflammatory cell activation. Control macrophages are rounded (panel a), macrophages are visible for antennary production upon LPS stimulation (panel c), and all experimental intervention groups have the ability to inhibit their activation (panel b, panel d, panel e), where the patch-carrying group is more potent than the empty patch group (panel f): p is less than 0.05.
FIG. 9 shows the effect of the film material on the secretion of inflammatory cell factors. In the figure, the PCR method is used for measuring the expression of inflammatory factors TGF-beta 1(a picture), IL-6(b picture) and TNF-alpha (c picture) of each experimental group, and the ELISA method is used for measuring the release of the inflammatory factors TGF-beta 1(d picture), IL-6(e picture) and TNF-alpha (f picture) of each experimental group. Each experimental group had the ability to inhibit the secretion of inflammatory factors, with the C/P group being more potent than the empty patch group: p is less than 0.05.
FIG. 10 shows that the patch material can inhibit fibrosis by inhibiting the release of inflammatory factors. In the figure, the recombinant TGF beta-1 induces the L929 cells to differentiate into myofibroblasts, the expression of alpha-SMA, collagen I and collagen III is obviously increased, the leaching solution of an empty load adhesive film and a load PLGA adhesive film in 48 hours cannot obviously reverse the activation, but the triamcinolone acetonide nude drug can inhibit the activation (a, c and e); whereas the 96h C/C and C/P groups of leachates were all able to significantly reverse fibroblast activation (panels b, d and f): p is less than 0.05.
FIG. 11 is a WB detection diagram, and both the no-load patch and the drug-loaded patch can inhibit the activation of fibroblasts induced by TGF beta-1.
Fig. 12 shows the effect of the patch material in inhibiting fibrosis by inhibiting the release of inflammatory factors. Grouping in the figure: lane 1 on the far left, and normal medium and LPS added to L929 fibroblasts; lane 2: adding unstimulated RAW264.7 macrophage supernatant and LPS into L929; lane 3: adding LPS to L929 to stimulate RAW264.7 supernatant for 24 hours; lane 4: adding LPS stimulated RAW264.7 supernatant cultured by no-load pad pasting material leachate into L929; lane 5: adding LPS stimulated RAW264.7 supernatant cultured by carrier membrane leaching solution into L929; lane 6: adding triamcinolone acetonide nude drug treated LPS stimulated RAW264 supernatant into L929), thereby proving that the film-sticking material can inhibit fibrosis by inhibiting the release of inflammatory factors.
FIG. 13 is the creation of dermal defect area on rat back (panel a), wound covering with adhesive film (panel b), covering with gauze (panel c) and fixing (panel d), 7 days later observation (panel e) and dissecting to analyze wound.
FIG. 14 is a 7 day time point immunohistochemical analysis of macrophage (CD68), neutrophil (MPO), TGF-. beta.1, and α -SMA expression from dissected wounds.
FIG. 15 shows that the level of expression of the macrophage marker CD68 in the wound surface of each experimental intervention group is lower than that in the control group (a panel); the neutrophil indicator MPO was also lower than the control (panel b); similarly, the proliferation indexes TGF-beta 1 (figure c) and alpha-SMA (figure d) are also lower; meanwhile, the inhibition of the C/C/P group on the proliferation capacity of macrophages, neutrophils and fibroblasts is stronger than that of the unloaded C/C patch group; *: p is less than 0.05.
FIG. 16 shows the detection of wound surface by different staining methods at 42 day time points. The combined triamcinolone acetonide/PLGA drug microsphere adhesive film (chitosan/collagen) acts on the wound surface together, and can effectively inhibit the thickness and hardness of scars.
Fig. 17 detection of wound conditions: triamcinolone acetonide, the no-load adhesive film and the drug-loaded adhesive film all inhibit the contractibility of wound scars, and the inhibition capacity of the drug-loaded adhesive film is stronger than that of the no-load adhesive film material (a picture); each experimental group is smaller than the control group on the scar thickness of the wound surface, and the hyperplasia inhibiting capacity of the drug-loaded adhesive film is stronger than that of the no-load adhesive film material (b picture); there was no statistical difference in the amount of fibroblasts in scar tissue after 6 weeks (panel c); *: p is less than 0.05.
Detailed Description
In order to make the invention more comprehensible, preferred embodiments are described in detail below with reference to the accompanying drawings:
as shown in figures 1-17, the invention provides a degradable stent for preventing and treating esophageal stenosis, which comprises a film-sticking material of genipin cross-linked chitosan collagen loaded with PLGA/triamcinolone acetonide sustained release microspheres and a degradable stent; the periphery of the degradable stent is provided with a genipin cross-linked chitosan collagen film material loaded with PLGA/triamcinolone sustained release microspheres. The main body material of the degradable stent comprises polylactic acid, and the degradable stent is woven by an electrostatic spinning weaving process. The degradable stent for preventing and treating esophageal stenosis provided by the invention can be applied to treatment of benign esophageal stenosis. The degradable stent for preventing and treating esophageal stenosis provided by the invention can be applied to treatment of esophageal ESD postoperative stenosis. The degradable stent for preventing and treating the esophageal stenosis provided by the invention can be applied to treatment of anastomotic stenosis after chest surgery esophageal surgery and scar contracture stenosis after esophageal chemical injury.
The invention takes polylactic acid material as a main body, prepares the esophagus degradable stent and a corresponding stent release device (shown in table 1) by an electrostatic spinning weaving process, and performs in-vitro experiments and animal experiments. In vitro degradation tests verified that the degradation time (disintegration) of the degradable stent was adjusted to 12 weeks while ensuring the radial supporting force (see tables 2 and 3). And then, carrying out animal experiments, and constructing an esophageal stenosis model simulating clinical objective and actual by carrying out circumferential esophageal mucosal stripping of 3cm length on 5cm of esophageal mucosa on cardia of an experimental animal by using an ESD method in advance by the applicant. The esophageal lumen stenosis of the experimental animal can be seen under endoscopic observation for 2-3 weeks after ESD operation, and only a 1-2mm gap is reserved. After the animal model is successfully established, the degradable stent is placed, and the patient is closely observed after the animal model is placed, so that the narrow section is expanded, the degradable stent is moved to the gastric cavity after about one week, and the narrow section is still unblocked (an endoscope can pass).
Animal experiment results of early-stage degradable stents suggest that the problems of traditional stents can be overcome by simply applying the degradable stents, but the stents are not narrowed after a narrow section is expanded for a period of time by the radial force of the stents, the degradable stents can shift about 7 days, and the in-situ time of certain stents is prolonged by methods of metal clip auxiliary fixation, stent shape and diameter improvement, weaving process optimization, surface friction force increase and the like in follow-up research, but the stent shift is finally inevitable. Once the stent is displaced from the original narrow section, the narrow section loses physical radial supporting force, but the time span of scar tissue hyperplasia of the wound surface is larger than the '7 days' of the stent for temporarily solving the problem of stenosis, so that the radial supporting force of the stent is not available, and the continuous hyperplasia of the scar tissue inevitably causes restenosis. Therefore, the stricture can be relieved only in a short time and the long-term effect cannot be achieved only by a physical method (stent) without starting from a molecular mechanism for inhibiting the hyperplasia of scar tissues, and the stricture is one of the root causes of repeated stricture but repeated restenosis of esophagus stricture in clinic. Thus, not only does physical radial force be required to support the stenosis, but a combination of drugs is required to inhibit fibrous tissue proliferation from the molecular mechanism to address the symptoms and root causes of post-ESD stenosis.
The invention successfully prepares the genipin cross-linked chitosan collagen film material loaded with PLGA/triamcinolone acetonide sustained release microspheres. The observation structure of an electron microscope and the detection of the drug concentration by a liquid-phase mass spectrometry prove that the genipin cross-linked chitosan collagen film loaded with PLGA/triamcinolone acetonide sustained release microspheres prepared by the invention can effectively release the triamcinolone acetonide for a long time (more than 3 months). In vitro cell experiments demonstrated that it inhibits the fibrosis of muscle cells by inhibiting the release of cytokines TNF- α, IL-6, TGF- β 1, etc., from inflammatory cells (macrophages, neutrophils, etc.), inhibiting α -SMA expression, preventing collagen deposition (fig. 3-12). Experiments on the repair of skin scars of small animals find that the scar repair cream has a definite tissue fibrosis inhibition effect on ESD wound surfaces within about one week and has medium-long term effects (figures 13-17).
If the drug carrier is applied in a combined manner, the esophageal stenosis is systematically prevented, the drug carrier film can be closely attached to the wound surface for 7 days to release drug effect, the problem of restenosis after 7 days of displacement of the degradable stent can be solved, and the effect of treating both symptoms and root causes can be expected to be achieved.
Examples
1. Animal model for establishing esophageal ESD postoperative stenosis
(1) A Shanghai white pig (15-20kg, male) is used, intubation anesthesia is performed after fasting for one day, and 5cm of esophageal mucosa on cardia is peeled off all around under endoscopic mucosa, and the longitudinal diameter is about 3 cm.
(2) And 3, observing the effect of the esophagoscope around ESD operation under the endoscope for 3 d, 7 d, 14 d, 21 d and 42d, and recording the time when the stenosis occurs and the diameter of the lumen. After about 2-3 weeks of ESD operation, esophageal stenosis is clearly defined, food residues can be seen under the endoscope, and a narrow pore of about 1-2mm can be seen after cleaning.
2. Degradable stent
(1) Compared with the material research at home and abroad and the clinical actual demand, the polylactic acid is selected as the main material of the degradable stent, and the stent is prepared by applying the electrostatic spinning weaving process.
(2) The characteristics of the specification and the shape of the stent, the weaving density and the like are designed by combining clinical requirements and experimental prediction, and the conveying device is designed, produced and prepared to provide experience for subsequent improvement.
(2.1) pre-experimental scaffold size information. See table 1:
TABLE 1 Stent size information
| Diameter of stent body | Diameter of flaring | Length of |
| 18mm | 23mm | 90mm |
(2.2) preparing an experimental bracket;
(2.3) preparing an experiment conveyor;
(3) and (3) carrying out contrast test on the three types of esophageal stents, and verifying the mechanical properties of the pre-experimental samples.
A Blockwise radial force tester (Shanghai medical instrument detection institute) is used for testing a clinically used A film-covered metal bracket, a B metal bare bracket and a sample pre-experiment bracket C of the project group.
And (3) testing conditions are as follows: the loading speed is 0.1mm/s, the testing temperature is 37 ℃, and the cycle times are 3 times. See table 2:
TABLE 2 comparison table of test results of three kinds of supports
| Sample number: | maximum applied external diameter (mm)/force value (N/mm) | Minimum application outside diameter (mm)/force value (N/mm) |
| A | 18.9/0.097 | 9.9/1.687 |
| B | 18.9/0.056 | 9.9/0.151 |
| C | 17.955/0.021 | 10.305/0.239 |
Conclusion of preliminary experiments: analysis from force values the degradable stent was inferior to the metallic peritoneal stent sample a, but the degradable stent sample C was superior to the metallic bare stent sample B. The degradable stent sample C is soft (the force value is small) in the initial stage, can change along with the change of the diameter of the esophagus and reduce the stimulation to the esophageal wall; the later stage can promote the holding power fast along with the pipe diameter reduces, satisfies the narrow treatment demand of esophagus.
(4) And (3) testing the accelerated degradation support performance:
degradation conditions are as follows: the test result shows that the radial force (the upper part and the lower part are both curved surface pressure heads, the flaring diameter of the support is 23mm, and the test stroke is 15mm) is prompted by the test result of accelerated degradation in artificial gastric juice at 55 ℃ for 7 days (simulating the actual degradation for about 6 weeks). See table 3:
TABLE 3 Change in mechanical Properties of scaffolds before and after degradation
Conclusion of preliminary experiments: the trial-produced degradable scaffold can provide satisfactory radial support in a 6-week simulated environment. The accelerated degradation is continued for 14 days and 21 days, and the stent is simulated to be placed in the body for 3-6 months, so that the stent is degraded and fragmented, and is in line with the expectation.
(5) Application of pre-experimental sample support in esophageal stenosis animal model experiment
Specifically, the animal model of esophageal stenosis observes a target narrow section by applying a degradable stent endoscope, and after a zebra guide wire passes through the narrow section, the zebra guide wire is guided by the guide wire and is placed into an expansion balloon, the narrow section is expanded to 10mm in advance, and the bleeding of a wound surface can be seen; after the operation, the stent can be seen to shift under the endoscope after about 7 days, but the endoscope in the target narrow section can still pass through smoothly, and the narrow section can be seen to be completely narrowed again after the stent is shifted for 2 weeks. The degradable stent can be seen to shift about 7 days after the operation, the esophageal lumen is slightly narrow, but the endoscope can completely pass through; the stent is observed to shift and fall into the gastric cavity under the endoscope, a large amount of digestive juice soaks the stent, the original esophageal stenosis part is narrowed again about 2 weeks after the stent is shifted, and a small amount of chyme cannot pass through the endoscope and can be seen.
Conclusion of animal experiments on degradable scaffolds in preliminary experiments: the stenosis can be temporarily solved by simply placing the degradable stent from the physical and mechanical aspect and immediately and progressively expanding the stenosis section to about 7 days, but then the stent is difficult to avoid shifting due to no compression action after the stenosis is relieved, and the original stenosis section is narrowed again soon after the stent is shifted.
3. Developing a film material with effect of inhibiting scar hyperplasia on molecular mechanism level
(1) Preparing PLGA/triamcinolone acetonide sustained release microspheres and a microsphere-loaded chitosan collagen film-pasting material; preparing PLGA/triamcinolone acetonide drug-loaded microspheres: the invention adopts a classical solvent volatilization method to prepare the drug-loaded microspheres, and adopts an orthogonal method to analyze the influence of different preparation conditions (emulsifier/rotating speed/volatilization time and the like) on the physicochemical properties (grain diameter/swelling capacity/degradation) and the sustained-release capacity of the microspheres.
Preparing a chitosan collagen film crosslinked by genipin: dissolving chitosan powder and atelocollagen in diluted acetic acid (2%) according to the ratio of (1: 1), adding cross-linking agent (0.1% genipin), adding the prepared drug-loaded microspheres, cross-linking at 37 deg.C for different time, freeze-drying, and titrating to neutrality.
The particle size of the PLGA/triamcinolone acetonide sustained release microspheres (as shown in figure 1) and the chitosan/collagen film structure (as shown in figure 2) loaded with the triamcinolone acetonide/PLGA sustained release microspheres and genipin crosslinking are observed through a light mirror and an electron microscope.
(2) PLGA/triamcinolone acetonide sustained release microspheres and determination of sustained release capacity of carrier ball framework
Due to the special acidic environment of the esophagus, the sustained release performance of the microspheres and the microsphere-loaded adhesive film on triamcinolone acetonide in different esophagus pH states is measured in vitro, and the drug sustained release capability of the microsphere-loaded adhesive film material in an in vivo environment is simulated.
As shown in fig. 3: putting the PLGA/triamcinolone acetonide sustained release microspheres and the carrier pasting film in phosphate buffer solutions with different pH values of 5, 6 and 7, selecting time points of 1, 3, 6, 10, 15, 22, 32 and 42 … … 92 to respectively exhaust the drug-containing buffer solution and replace a new buffer solution, measuring the release dosage at different time points by HPLC (high performance liquid chromatography), drawing a sustained release curve, and comparing the influence of different pH values and the influence of the stent on the sustained release capacity of the microspheres. Wherein, a figure is a liquid chromatogram drug release curve chart of PLGA/triamcinolone acetonide drug-loaded microspheres and b figure is a drug release curve chart of the chitosan/collagen adhesive film loaded with the triamcinolone acetonide/PLGA microspheres.
(3) In vitro cell experiments
The direct influence of the film material on inflammatory cells and fibroblasts:
mouse macrophage (Raw264.7) and fibroblast (L929) are respectively cultured to carry out CCK8 cell activity experiments, and Western blot detection is assisted to prove that the film material loaded with the drug microspheres can promote the activity of inflammatory cells and inhibit the activity of the fibroblast.
As shown in fig. 4, 5, 6, and 7, the groups in the drawings represent the following: NC blank culture medium, 2.5mg/ml material leaching solution of C/C low chitosan/collagen pad pasting, 5mg/ml material leaching solution of C/C high chitosan/collagen pad pasting, 2.5mg/ml material leaching solution of C/C/P low chitosan/collagen/PLGA drug-loaded microsphere pad pasting, and 5mg/ml material leaching solution of C/C/P high chitosan/collagen/PLGA drug-loaded microsphere pad pasting.
The C/C group and C/C/P group in FIG. 4 can promote the proliferation of inflammatory macrophages and inhibit cell death (see a); both C/C and C/C/P groups inhibited cell proliferation and death in fibroblasts, but the inhibition of proliferation was more pronounced in comparison (see b).
The WB assay in FIG. 5 further demonstrated that the C/C and C/C/P groups promoted inflammatory cells and inhibited fibroblasts compared to the control group.
FIG. 6 is a graph showing the effect of the patch material on the activity of murine macrophages (Raw264.7) which are inflammatory cells; in the figure, the macrophage activity of each group of 24h is not different (as a picture), but the macrophage promoting effect can be achieved in 48h of the C/C/P high group (as b picture), the macrophage calcein marks of the C/C group and the C/C/P group are more than those of the control group (as C picture), and in contrast, the PI indexes are lower than those of the control group (as d picture), and the ratio of the two is as follows: p is less than 0.05.
FIG. 7 is a graph showing the effect of the patch material on the activity of fibroblasts (L929); in the figure, the C/C/P high group has the effect of inhibiting fibroblasts after 24h (shown as a figure), 48h of each experimental group has the effect of inhibiting the C/C low group compared with the control group, and the effect of inhibiting the C/C/P high group is stronger than that of inhibiting the C/C/P low group (shown as a figure b); the calcein markers of the fiber cells of the C/C group and the C/C/P group are lower than those of the control group (such as a C picture), and on the contrary, the calcein markers of the fiber cells of the PI group are higher than those of the control group (such as a d picture): p is less than 0.05
Secondly, the influence of the sticking film on the activation of inflammatory cells and secretion factors is further analyzed;
the mouse macrophage RAW264.7 after being stimulated and activated by LPS can be changed from a round shape into an antennal cell and secretes a large amount of fibrosis-causing factors, but the no-load adhesive film and the loading ball adhesive film can inhibit the activation of the mouse macrophage RAW264.7 and reduce the secretion of the cell factors (TNF-alpha, IL-6 and TGF-beta 1) for promoting the fibrosis. See fig. 8, 9:
in FIGS. 8 and 9, the group was con without stimulation, LPS group was stimulated for 24 hours, LPS + group C/C LPS + medium containing 5mg/ml chitosan/collagen patch leachate was co-stimulated for 24 hours, LPS + group C/C/P LPS + medium containing 5mg/ml chitosan/collagen/PLGA patch leachate was co-stimulated for 24 hours, and LPS + group ta LPS + medium containing the same triamcinolone acetonide nude drug was co-stimulated for 24 hours.
As shown in fig. 8, the macrophages in the control group are rounded (see a), the macrophages are observed to produce tentacles when stimulated by LPS (see c), and all experimental intervention groups have the ability to inhibit their activation (see b, d, e), wherein the patch-loaded membrane group is more potent than the empty patch group (see f): p is less than 0.05
As shown in FIG. 9, the expression of the inflammatory factors TGF-. beta.1 (as shown in panel a), IL-6 (as shown in panel b) and TNF-. alpha. (as shown in panel c) was measured for each experimental group by PCR method, and the release of the inflammatory factors TGF-. beta.1 (as shown in panel d), IL-6 (as shown in panel e) and TNF-. alpha. (as shown in panel f) was measured for each experimental group by ELISA method. Each experimental group had the ability to inhibit the secretion of inflammatory factors, with the C/P group being more potent than the empty patch group: p is less than 0.05
③ the action of the adhesive film on the activation and inhibition of fibroblasts:
fibroblast activation into myofibroblasts is an important cellular event occurring in fibrosis, and the process is that various inflammatory factors (especially TGF beta-1) act on fibroblasts to cause the increase of the framework protein alpha-SMA, so that the cells have a larger contraction phenotype, and secrete a large amount of collagen to cause scar formation. Further cell experimental analysis concluded that the patch material can inhibit fibrosis by inhibiting the release of inflammatory factors. As shown in fig. 10, 11 and 12:
FIG. 10 shows that recombinant TGF beta-1 induces L929 cells to differentiate into myofibroblasts, the expression of alpha-SMA, collagen I and collagen III is remarkably increased, the leaching solution of an empty load pad pasting and a load PLGA pad pasting in 48h cannot remarkably reverse the activation, but the triamcinolone acetonide nude drug can inhibit the activation (such as a picture a, a picture c and a picture e); whereas the 96h C/C and C/P groups of leachate both significantly reversed fibroblast activation (e.g. panels b, d and f): p is less than 0.05.
FIG. 11 shows that the no-load adhesive film and the drug-loaded adhesive film can inhibit the activation of fibroblast induced by TGF beta-1 through WB detection.
Fig. 12 is a WB assay to further investigate whether the patch reduces the release of inflammatory factors by inhibiting the activation of inflammatory cells, thereby inhibiting the fibrotic process. The supernatant (containing cell factors) of the RAW264.7 cells stimulated by LPS is used for stimulating the L929 cells, and as the fact that the leachate in 48h can not directly and obviously inhibit the activation of the fibroblasts is proved, 48h is selected as a node to eliminate the direct interference effect of a film pasting material on the activation of the fibroblasts. In the figure, the grouping is from left to right, the leftmost side is set as lane 1, and the normal medium and LPS are added into the L929 fibroblast; lane 2, unstimulated RAW264.7 macrophage supernatant and LPS was added to L929; lane 3, RAW264.7 supernatant stimulated for 24 hours with LPS in L929; lane 4, addition of LPS-stimulated RAW264.7 supernatant cultured with empty-loaded membrane-bound material leachate to L929; lane 5, LPS-stimulated RAW264.7 supernatant cultured with pellet-loaded membrane-applied leachate was added to L929; lane 6, the LPS-stimulated RAW264 supernatant treated with triamcinolone acetonide was added to L929, thereby demonstrating the effect of the patch material in inhibiting fibrosis by inhibiting the release of inflammatory factors.
(4) Dermal defect experiment of rat
(4.1)
At the 7 day time point, the patch material studied inhibited the expression of macrophages (CD68), neutrophils (MPO), TGF-. beta.1, and alpha-SMA on a molecular mechanistic level. See fig. 13, 14, 15:
FIG. 13 is the dermal defect area (fig. a) established on the back of rat, wound is covered with adhesive film (fig. b), then covered with gauze (fig. c) and fixed (fig. d), observed after 7 days (fig. e) and dissected to analyze the wound.
FIG. 14 is a 7 day time point immunohistochemical analysis of macrophage (CD68), neutrophil (MPO), TGF-. beta.1, and α -SMA expression from dissected wounds.
FIG. 15 shows that the level of expression of the macrophage marker CD68 in the wound surface of each experimental intervention group is lower than that in the control group (a panel); the neutrophil indicator MPO was also lower than the control (see panel b); similarly, the proliferation indexes of TGF-beta 1 (shown as a figure c) and alpha-SMA (shown as a figure d) are also lower; meanwhile, the inhibition of the C/C/P group on the proliferation capacity of macrophages, neutrophils and fibroblasts is stronger than that of the unloaded C/C patch group; *: p is less than 0.05.
(4.2) and 42 days, detecting the scar condition of the wound surface: the combined triamcinolone acetonide/PLGA drug microsphere adhesive film (chitosan/collagen) acts on the wound surface together, and can effectively inhibit the scar thickness and the scar hardness in a long term. See fig. 16, 17:
FIG. 16 shows the detection of wound surface by different staining methods at 42 day time points.
Fig. 17 shows that triamcinolone acetonide, the no-load adhesive film and the drug-loaded adhesive film all inhibit the shrinkage of wound scars, and the inhibition capacity of the drug-loaded adhesive film is stronger than that of the no-load adhesive film material (as shown in a picture); each experimental group is smaller than the control group on the scar thickness of the wound surface, and the medicine-carrying adhesive film has stronger proliferation inhibition capability than the no-load adhesive film material (as a picture b); there was no statistical difference in the amount of fibroblasts in scar tissue after 6 weeks (see panel c); *: p is less than 0.05.
(5) The combined application comprises the following steps:
(1) preventive use: the esophagus ESD postoperative of the animal model is immediately covered by the drug-carrying adhesive film, and then the drug-carrying adhesive film is pressed and fixed by the degradable bracket, so as to prevent the postoperative stenosis of the esophagus ESD.
(2) Therapeutic use: the drug-loaded full-coated degradable stent containing the triamcinolone acetonide microspheres is prepared and applied to animal experiments.
With the popularization of the endoscopic diagnosis and treatment technology, esophageal tumor lesions can be treated more and more through endoscopic submucosal dissection, but the problem of esophageal stenosis caused by the esophageal tumor lesions becomes a main contradiction which affects the quality of life of patients after operation, and the esophageal tumor lesions are not healed for a long time, and in turn, the deep development and popularization of endoscopic diagnosis and treatment are also affected. Meanwhile, benign strictures of the esophagus, such as benign strictures of an anastomotic stoma after esophageal cancer operation in thoracic surgery, inflammatory strictures caused by chemical burns of the esophagus due to mistaken eating of strong acid and strong base, which are more common in rural areas, are also problems to be solved clinically.
For patients with benign stricture of esophagus, such as stricture after ESD operation, after the guide wire is used for passing through the narrow section, the guide wire under the endoscope is guided through the conveyor to release the full-film degradable stent in the narrow section. In addition, the novel degradable stent can be used for preventive use, and can be immediately used in an ESD wound area to act on a possibly narrow section after an ESD operation.
When the endoscope reaches a target area, the guide wire is kept, the full-coated degradable stent is placed on a target narrow section or a wound surface which can be narrow through a conveyor under the guide of the guide wire, and the endoscope is withdrawn. Followed by regular follow-up visits.
While the invention has been described with respect to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention. Those skilled in the art can make various changes, modifications and equivalent arrangements, which are equivalent to the embodiments of the present invention, without departing from the spirit and scope of the present invention, and which may be made by utilizing the techniques disclosed above; meanwhile, any changes, modifications and variations of the above-described embodiments, which are equivalent to those of the technical spirit of the present invention, are within the scope of the technical solution of the present invention.
Claims (5)
1. A degradable stent for preventing and treating esophageal stenosis is characterized in that: comprises a film pasting material and a degradable stent of genipin cross-linked chitosan collagen loaded with PLGA and triamcinolone acetonide sustained release microspheres; the periphery of the degradable stent is provided with a genipin cross-linked chitosan collagen film material loaded with PLGA/triamcinolone sustained release microspheres.
2. The degradable stent for preventing and treating esophageal stenosis of claim 1, wherein: the main body material of the degradable stent comprises polylactic acid, and the degradable stent is woven by an electrostatic spinning weaving process.
3. The use of the degradable stent of claim 1 for preventing and treating esophageal stenosis for treating benign stenosis of esophagus.
4. The use of the degradable stent of claim 1 for preventing and treating esophageal stenosis for treating esophageal post-ESD stenosis.
5. The use of the degradable stent of claim 1 for preventing and treating esophageal stenosis for treating anastomotic stenosis after chest surgery and esophageal chemical injury and scar contracture stenosis after esophageal chemical injury.
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