Microorganism in-situ synthesis integrated covered stent as well as preparation method and application thereof

Abstract

The invention provides a microorganism in-situ synthesis integrated covered stent as well as a preparation method and application thereof, and belongs to the technical field of tissue engineering stent materials. The surface of a metal support is coated with CaO2 nano-particles through photocuring hydrogel, the metal support is fixed in a customized reactor filled with fermentation liquor, oxygen is slowly released through slow hydrolysis of the CaO2 nano-particles, gluconacetobacter xylinum is driven to be colonized and proliferated on the surface of the metal support, and nano-cellulose is gradually synthesized from thalli in situ. And finally, coating the metal mesh of the stent with the nanocellulose at 360 degrees, and synthesizing the integrated stent of the stent and the covering film in situ. The covered stent is subjected to 10000 times of cyclic compression, the stent structure is stable, the stress of the bare metal stent and the covered stent is 3.5 N when the stent is compressed to 90%, and the mechanical stability reaches 99.999%. In addition, the integrated covered stent has the characteristics of large-capacity drug loading, high biocompatibility and high in-vivo safety.

Description

Technical Field

[0001] The present invention belongs to the technical field of tissue engineering scaffold materials, and particularly relates to a microorganism in-situ synthesis integrated film-covered stent, a preparation method thereof and an application thereof. Background Art

[0002] The treatment strategy of using artificial tubular stents to replace diseased cavities is widely used in cavities such as cardiovascular and cerebrovascular, esophagus, trachea, intestine, biliary tract, urethra, etc. Most cavities have an outer skin support with superior mechanical properties and an endothelial system with biological functions to achieve corresponding physiological functions. Currently, except for drug-coated small-diameter vascular stents, there are no other biofunctional stents on the market. Among commercial stents, plastic tubular stents are limited in long-term implantation and the preparation of large-diameter stents due to mechanical properties and rejection problems. Silicone stents have a high migration rate and high cost, which hinder their large-scale application. Metal stents have high deformation force, fatigue resistance, and low migration rate, are suitable for minimally invasive placement and removal, and their mechanical properties are irreplaceable in maintaining the shape of the cavity. Improving the film-covered scheme based on metal stents to achieve biological functions is currently the most transformative path.

[0003] Film-covered stents replacing surgical operations are becoming increasingly important in modern medicine. However, existing film-covered stents generally have problems such as easy film shedding, poor compliance, and poor tissue compatibility, which seriously hinder the development of tissue engineering scaffolds.

[0004] Currently, the film-covered stent products developed based on metal stents clinically mainly include implants such as Zenith of Cook Medical, Talent, Endurant, and AneuRx of Medtronic, Excluder of Gore, and Vanguard of Boston Scientific. The film-covered materials mainly include polytetrafluoroethylene (PTFE), polyester fiber, polyester, etc. These synthetic materials only serve as a physical isolation effect and lack biological functions. The research on materials used in biofunctional metal stents under research focuses on developing new biomaterials such as hydrogels and decellularized matrices, which are mostly applied to the regeneration of single tissues such as bone. The combination method with tubular metal stents mostly relies on simple surface attachment and wrapping. This type of film-covered construction scheme has the problem of mismatched mechanical properties after film formation, which is extremely likely to cause film shedding and re-blockage of the cavity. The preparation cost of decellularized matrices is high, and they cannot provide an exact match in terms of the size and mechanical properties of the patient's specific cavity, and there is a risk of in vivo collapse. The integration of the film and the metal stent determines the long-term safety of the stent implanted in the body and is a decisive factor in the clinical transformation of film-covered stents.

[0005] Therefore, existing film-covered stents generally have problems such as easy film shedding, poor compliance, and poor tissue compatibility, which seriously hinder the development of tissue engineering scaffolds.

[0006] 3D printing can utilize composite materials to construct multi-layered tubular scaffolds. However, limited by the low toughness of existing materials, there is no complete alternative to metal scaffolds for applications requiring high stress in plastic deformation. The construction of coated scaffolds by electrospinning has been reported in skin repair and urethral repair. However, the limited choice of electrospinning materials, the poor mechanical stability of natural materials, the poor biocompatibility of synthetic materials, and the inherent defects such as the difficulty in mass manufacturing of hybrid materials affect their applications and make it difficult to achieve additive manufacturing based on metal frameworks. Therefore, there is an urgent need for new solutions to construct an integrated coated metal scaffold to achieve biological functions.

[0007] However, there is currently no customizable coating solution based on metal scaffolds. This may be related to the combination method of other materials with metal scaffolds. The surface contact area is too small to generate sufficient adhesion, or the generation process cannot be customized. This will also lead to adverse clinical outcomes due to the interference of the over-covered part with normal endothelial function.

[0008] Nanocellulose (Bacterial nanocellulose, BNC), as a new type of biomaterial, has been widely studied and applied in in vivo implants and drug carriers. It has a 3D ultrafine fiber network structure, good mechanical properties and biocompatibility, and the production process is simple and customizable. Cherng et al. successfully used BNC materials as a matrix to maintain the stemness of epithelial cells and induce differentiation. Baoxiu Wang et al. constructed a bacterial nanofiber-based porous scaffold and successfully used it for urethral repair. BNC has a high tensile strength and excellent flexibility, but lacks support and it is difficult to maintain a tubular morphology without external support. Its tensile strength comes from its unique fiber network structure and strong hydrogen bond interactions, which enable BNC to be formed into any shape while maintaining the integrity of its surface. At the same time, thanks to the unique production mode of progressive formation of bacteria, the implants in the BNC membrane production environment can be completely wrapped by the BNC membrane and obtain the same tensile strength, that is, the tensile strength does not change drastically and break due to the interface change at the material edge, but the BNC membrane is completely continuously covered to obtain a uniform tensile strength. At the same time, the BNC membrane maintains its fiber network structure at the micro level and maintains its flexibility. This is significantly different from the current hot pressing method and sewing method of coated materials. The BNC and metal hybrid materials mostly use BNC frameworks and different metal fillers to obtain additional electrical and thermal conductivity, and the method of coating BNC on a metal framework has not been reported.

[0009] However, there are still difficulties in preparing an integrated coated metal scaffold. In particular, how to achieve the controllable production of coated metal scaffolds, and prepare a BNC-coated integrated metal scaffold with low rejection, low production cost, and high customization has become a technical problem to be solved urgently. Summary of the Invention

[0010] The present invention is to solve the above technical problems, and thus provides a microbial in-situ synthesis integrated film-covered stent and its preparation method and application. The technical object of the present invention is to solve the problems that the existing solutions cannot successfully prepare an integrated film-covered metal stent, and it is difficult to prepare a BNC film-covered integrated metal stent with low rejection, low production cost, and high customization.

[0011] In order to achieve the above technical object, the technical solution adopted by the present invention is as follows:

[0012] The present invention first provides a preparation method for a microbial in-situ synthesis integrated film-covered stent, including the following steps:

[0013] (1) Mix CaO2 nanoparticles with a GelMA hydrogel solution, and then immerse the metal stent in the hydrogel solution; after taking it out, cure it under ultraviolet light to obtain a metal stent loaded with CaO2 nanoparticles;

[0014] (2) Inoculate Gluconacetobacter xylinus into a fermentation medium, and shake and culture to obtain an activated bacterial culture solution;

[0015] (3) Fix the metal stent loaded with CaO2 nanoparticles between two silicone tubes with different inner and outer diameters to prepare a reactor, inoculate the fermentation broth filled with the activated bacteria obtained in step (2) into the sterilized reactor, and place it at 30 °C for static culture for 14 days to obtain a nanocellulose film-covered stent. After removing the reactor, purify the nanocellulose film-covered stent to obtain the integrated film-covered stent.

[0016] The method of the present invention slowly releases oxygen through the prepared metal stent loaded with CaO2 particles, drives the bacteria to colonize around the metal stent, and gradually generates nanocellulose, realizing 360-degree wrapping of the metal network by nanocellulose, thereby successfully constructing an integrated film-covered stent. By mixing CaO2 nanoparticles with GelMA hydrogel, the present invention constructs a metal stent coated with CaO2 nanoparticles, which has the effect of releasing oxygen, thereby achieving the effect of driving bacteria to colonize around the metal stent and realizing an integrated BNC film-covered stent. Compared with the later physical integration of other membranes and stents, this integrated method will not cause the phenomenon of membrane-stent separation.

[0017] On the other hand, a small amount of GelMA hydrogel and CaO2 nanoparticles in the coating process only play the role of releasing oxygen. As the fermentation process progresses, the above materials will be completely degraded within 5 days. Therefore, the finally obtained is a pure BNC film-covered stent, which does not contain the components of GelMA hydrogel and CaO2 nanoparticles; this is essentially different from the composites of GelMA hydrogel or other nanoparticles and bacterial cellulose.

[0018] The integrated BNC-coated stent prepared by the method of the present invention has high integrated stability. After being compressed 10,000 times, it still maintains the characteristic of the integration of the membrane and the stent. However, when attempting to use a method not driven by oxygen, that is, directly placing the metal stent in a mold for fermentation to prepare the BNC-coated stent, it is easy to produce the phenomenon of separation between the membrane and the stent after taking it out after fermentation, and it is difficult to achieve integration.

[0019] The research of the present invention shows that in vitro, the coated stent has good cell compatibility and can achieve effective loading and slow release of drugs. At the same time, it can achieve interventional targeted release in vivo, with low damage and low inflammatory response to airway mucosa. Further, the present invention realizes the asymmetric coated drug loading design on the nitinol tracheal stent based on the 3D printing template, which not only retains the supporting ability and deformation ability of the metal stent, but also can effectively delimit the coated range, realize customized coating within a fixed range, and achieve precise release in the delimited area after drug loading.

[0020] Therefore, the method of the present invention has the following advantages:

[0021] (1) The existing coated stents cannot prepare an integrated coated stent, and the membrane is easy to fall off and separate from the metal stent. However, the method of the present invention can well prepare an integrated coated stent without the separation and falling off of the membrane;

[0022] (2) The mechanical properties and mechanical stability of the coated stents prepared by the existing methods are not good, while the mechanical stability of the integrated coated stent obtained by the method of the present invention is extremely excellent;

[0023] (3) The existing methods directly prepare the coated stent by microbial fermentation. As shown in the comparative example, this method has the phenomenon of separation between the stent and the membrane; while the method of the present invention combines the oxygen-driven method and successfully prepares an integrated coated stent, and the integrated preparation of the coated stent is successfully realized through this method.

[0024] Furthermore, the mass-volume concentration of the GelMA hydrogel solution in step (1) is 7.5% (m / v), and the addition amount of the CaO2 nanoparticles in step (1) is 0.01 - 1% (m / v) in terms of mass-volume concentration.

[0025] Furthermore, the metal stent in step (1) includes any one of a nitinol stent, a cobalt-chromium alloy, a 316L stainless steel stent, a nickel stent, a tantalum stent, a magnesium stent, and a zinc stent.

[0026] Furthermore, the curing in step (1) is carried out by irradiating with ultraviolet light of 365 nm for 5 minutes.

[0027] Furthermore, the formula of the fermentation medium described in step (2) is: 100 g / L D-fructose, 5 g / L peptone, and 3 g / L yeast extract, with a pH of 5.0.

[0028] Furthermore, the sources of the Gluconacetobacter xylinus described in step (2) include the strains preserved on slants, the strains preserved in cryopreservation solutions, and the bacterial cellulose membranes with strains. The inoculation amount of the strains preserved on slants is 1 - 2 loops, the inoculation amount of the strains preserved in cryopreservation solutions is 1 - 5% (v / v), and the inoculation amount of the bacterial cellulose membranes with strains is 1 - 5 pieces of the bacterial cellulose membranes with a size of 1 - 5 mm. 3 of the bacterial cellulose membranes.

[0029] Furthermore, the cultivation in the fermentation medium in step (2) is carried out by shaking cultivation at 30 °C and 120 rpm, and the static cultivation is carried out by static cultivation in a constant temperature and humidity incubator at 30 °C for 7 days.

[0030] Furthermore, the inner diameter and outer diameter of the two silica gel tubes described in step (3) are adjustable sizes, and the inner diameter and outer diameter size ranges of the inner and outer silica gel tubes are 1.5 - 30 mm * 2 - 35 mm, and the inner diameter difference between the outer silica gel tube and the inner silica gel tube is 0.5 - 10 mm.

[0031] The second object of the present invention is to provide an integrated covered stent prepared by the method described above.

[0032] The third object of the present invention is to provide the application of the integrated covered stent described above in the preparation of tissue engineering scaffold materials.

[0033] The beneficial effects of the present invention are as follows:

[0034] In the present invention, CaO2 nanoparticles are coated on the surface of a metal stent through a photocurable hydrogel and fixed in a customized reactor filled with fermentation broth. The CaO2 nanoparticles slowly hydrolyze to continuously release oxygen, driving the colonization and proliferation of Gluconacetobacter xylinus on the surface of the metal stent. The bacteria gradually synthesize nanocellulose in situ, and finally the nanocellulose wraps the metal grid of the stent 360 degrees, and an integrated stent with a stent and a film is synthesized in situ. By performing live-dead bacteria staining on the film-covered stent during the synthesis process, it was determined that the bacteria colonized in situ around the metal stent under the drive of oxygen; the film-covered stent underwent 10,000 cycles of compression, and the stent structure was stable. When compressed to 90%, the stress of the bare metal stent and the film-covered stent was 3.5 N, and the mechanical stability reached 99.999%. After implanting the film-covered stent into the trachea of New Zealand white rabbits for 2 weeks, the tracheal patency rate was 100%, the tracheal epithelial cells in the film-covered area survived 100%, and there was no infiltration of inflammatory cells and goblet cell metaplasia. Through 3D printing modeling of clinical data, in a porcine trachea simulation experiment, it was shown that a customized film-covered stent loaded with FITC-PTX could achieve precise drug delivery. The integrated film-covered stent constructed by in-situ microbial synthesis of nanocellulose has the characteristics of high drug loading capacity, high biocompatibility, and high in-vivo safety, and is a very promising film-covered stent platform that can be applied to various in-vivo cavity interventional therapies. Description of the Drawings

[0035] Figure 1 It is a schematic diagram for the preparation of the integrated film-covered stent of the present invention.

[0036] Figure 2 It is for the preparation and physicochemical characterization of a stent loaded with CaO2 nanoparticles; A: Schematic diagram of the preparation process, B: Macroscopic morphology of the metal stents coated with GelMA and GelMA loaded with CaO2 nanoparticles respectively and the microscopic morphology observed by SEM; C: Element distribution maps (Ca / Ti / Ni / C / O / Merge) of GelMA and GelMA loaded with CaO2 nanoparticles by Mapping and element content statistical charts.

[0037] Figure 3 It is for the preparation and physicochemical characterization of a bare metal stent and a BNC film-covered stent; A: Macroscopic morphology during the in-situ microbial synthesis process of the BNC film-covered stent and the live-dead bacteria distribution map observed by fluorescence microscopy; B: Microscopic morphology diagram of the purified BNC film-covered stent; C: Element distribution maps (Ti / Ni / C / O / Merge) of the bare metal stent and the BNC film-covered stent by Mapping and D: Element content statistical charts.

[0038] Figure 4For cytocompatibility evaluation; A: Schematic diagram of evaluating cytocompatibility using extraction solution and co-culture; B: Cytotoxicity test of the extraction solution of the BNC-coated stent at 1, 3, and 7 days; C: Results of cytoskeleton fluorescence staining (F-actin / DAPI) after co-culture on the surface of the BNC membrane for 12, 24, and 48 h; D: Schematic diagram of the cytocompatibility test of the cell extraction solution of the PTX-loaded BNC-coated stent; E: Total amount of PTX released from the BNC membrane incubated with solutions of different PTX concentrations; F: PTX release curves with different drug loadings; G: Cytotoxicity of the stent with different PTX drug loadings.

[0039] Figure 5 For the preparation process and application of the asymmetric coated stent.

[0040] Figure 6 For the mechanical property evaluation of the BNC-coated stent; A: Stress-strain curve of the pure BNC membrane; B: Macroscopic view of the compression-release of the bare metal stent and the BNC-coated stent; C: Curves of the change in distance with time and the change in the force with time when the bare metal stent and the BNC-coated stent are compressed by 2, 4, 6, and 7 mm respectively; D: Curves of the change in the force with the compression distance when the bare metal stent and the BNC-coated stent are compressed by 2, 4, 6, and 7 mm respectively; E: Statistics of the average force for each distance when the bare metal stent and the BNC-coated stent are compressed by 2, 4, 6, and 7 mm respectively; F-G: DSA observation of the compression and release of the stent when the bare metal stent and the BNC-coated stent are placed in the rabbit trachea.

[0041] Figure 7 For: A) HE / PAS / MASSION staining and β-tubulin and CD45 fluorescence staining of rabbits implanted with bare metal stents and drug-loaded / non-drug-loaded coated stents for 2 weeks; B) Differences in the two immunostainings of the three different treatment methods; C) Statistical chart of the β-tubulin / CD45 fluorescence signal intensity of the bare metal stent, drug-loaded / non-drug-loaded fully coated stents.

[0042] Figure 8 For HE / PAS / MASSION staining and β-tubulin and CD45 fluorescence staining of rabbits 1 day, 1 week, and 2 weeks after implantation of bare metal stents and drug-loaded / non-drug-loaded coated stents. An indentation of the model stent can be seen at the contact surface between the stent and the mucosa. PAS staining shows an increase in the goblet cell model, but less CD45 staining on the surface. β-tubulin staining shows that the mucosa remains viable.

[0043] Figure 9 For the performance test of the integrated coated stent of the present invention.

[0044] Figure 10 For pictures of the coated stent of the present invention before compression (A) and after 10,000 compressions (B). Detailed implementation manners

[0045] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be specifically described below in combination with embodiments. It is necessary to point out that the following embodiments are only used to explain and illustrate the present invention, and are not used to limit the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above-mentioned inventive content still fall within the protection scope of the present invention.

[0046] Example 1

[0047] I. Experimental materials and methods

[0048] 1. Experimental reagents and materials

[0049] Unless otherwise specified, all chemical reagents in this embodiment are purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Tryptone, yeast powder and D-fructose are of the Adamas brand and are purchased from Shanghai Titan Scientific Co., Ltd. (Shanghai, China). The Komagataeibacter xylinus (K. xylinus) that produces bacterial nanocellulose (BNC) comes from the laboratory of Donghua University, and the number of the China General Microbiological Culture Collection Center is 1186. The human bronchial epithelial cell line HBE16 cells are purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). Fetal bovine serum (FBS), high-glucose DMEM medium, double antibiotics (10000 U / mL penicillin, 10000 μg / mL streptomycin) and 0.25% Trypsin-EDTA (1×) are all of the Gibco brand and are purchased from Shanghai Titan Scientific Co., Ltd. (Shanghai, China). The cell counting kit-8 (CCK-8) is purchased from Shanghai Beyotime Biotechnology Co., Ltd. (Shanghai, China). New Zealand white rabbits (male, about 4 kg) are purchased from Shanghai SLAC Laboratory Animal Co., Ltd. (SLAC, Shanghai, China). Nitinol metal stents with outer diameters of 8 mm and 12 mm are from Nanjing MicroPort Medical Science and Technology Co., Ltd. The live / dead bacteria staining kit (SYTO / PI) is purchased from Yeasen Biotechnology (Shanghai) Co., Ltd.

[0050] 2. Preparation of the CaO2 nanoparticle-coated metal stent (see Figure 1 )

[0051] (1) Preparation of GelMA and CaO2 nanoparticles

[0052] The synthesis method of GelMA hydrogel refers to the method in the existing literature. That is, 10 g of gelatin is weighed and added to 100 mL of PBS, heated and dissolved at 60 °C until clear, 4 mL of MA (methacrylic acid) is added at a rate of 250 μL / min with a micropump, stirred in the dark, and reacted at 60 °C for 2 h. Then 100 mL of PBS is added to terminate the reaction, and the precipitate is removed by centrifugation at 7000 rpm for 15 min. Dialysis is carried out at 37 °C for 3 - 4 days using a dialysis bag with a molecular weight cut-off of 8000 - 14000 Da, and then freeze-dried to obtain the freeze-dried GelMA sample for standby.

[0053] (2) Preparation and characterization of metal stent coated with CaO2 nanoparticles

[0054] Prepare 4 mL of 7.5% (m / v) GelMA hydrogel, take 0.04 g of CaO2 nanoparticles and resuspend them evenly in the hydrogel. After the metal stents are immersed in pure GelMA hydrogel and GelMA hydrogel containing CaO2 nanoparticles respectively and then taken out, they are cured by UV light at 365 nm for 5 min to obtain a metal stent wrapped with pure hydrogel (denoted as Gel-Stent) and a metal stent loaded with CaO2 nanoparticles (denoted as CaO2-Gel-Stent). The metal stent loaded with CaO2 nanoparticles (CaO2-Gel-Stent) is placed in water, and the generation of bubbles is observed, which is the production of oxygen. After the stent is freeze-dried, the microscopic morphology is observed by SEM and the element distribution and semi-quantitative element content are observed by Mapping.

[0055] 3. Construction of BNC integrated covered stent (see Figure 1 )

[0056] (1) Activation of bacteria and preparation of BNC membrane

[0057] Prepare the fermentation medium: 100 g / L D-fructose, 5 g / L peptone and 3 g / L yeast extract, adjust the pH to about 5.0, sterilize at 115 °C under high temperature and high pressure for 30 min. After standing at room temperature, the colonies of Gluconacetobacter xylinus are inoculated into the fermentation medium, and cultured with shaking at 30 °C and 120 rpm. When fine flocs can be seen with the naked eye, the fermentation broth full of activated bacteria is obtained for standby. The fermentation broth of the activated bacteria is inoculated into a 24-well plate, 1 mL per well, and placed in a constant temperature and humidity incubator at 30 °C for static culture for 7 days to obtain the BNC membrane.

[0058] (2) Assembly and sterilization of the reactor

[0059] Fix the metal stent loaded with CaO2 nanoparticles between two silicone tubes with inner and outer diameters of 9 * 10 mm and 6 * 7 mm respectively, and seal and fix both ends with silicone stoppers. After sterilization at 121 °C for 20 min under high temperature and high pressure, it is placed in a laminar flow cabinet for standby.

[0060] (3) Cultivation of the integrated film-coated stent and observation of the distribution of bacteria

[0061] The fermentation broth filled with activated bacterial strains was inoculated between the two silica gel tubes of the sterilized reactor to obtain a culture system, which was placed in a constant temperature and humidity incubator at 30 °C for static cultivation. After 3 days of cultivation, the macroscopic morphology of the BNC film-coated stent between the two silica gel tubes of the reactor was observed and recorded. The reactor was removed, and the bacteria on the film-coated stent were stained with a live / dead bacteria staining kit (SYTO / PI), and the distribution of bacteria on the film-coated stent was observed using a fluorescence microscope.

[0062] (4) Purification of the integrated film-coated stent and observation of its morphology

[0063] After the culture system was placed in a constant temperature and humidity incubator at 30 °C for static cultivation for 14 days, the reactor was removed, and the excess culture medium was rinsed off with deionized water. It was placed in a 1% (m / v) NaOH solution and boiled in alkali at 80 °C for 6 h, and the alkali solution was replaced every two hours. Subsequently, it was treated with ultrapure water at 80 °C for 48 h, and the ultrapure water was repeatedly replaced until the pH reached neutral. Finally, it was placed in a large amount of ultrapure water and treated at 121 °C for 20 min for 5 times, and the ultrapure water was replaced clean each time to remove endotoxins, obtaining a purified BNC film-coated stent with the endotoxin content meeting the implant standard for class III medical devices. The BNC membrane was purified in the same way. The purified BNC film-coated stent was immersed in ultrapure water, frozen at -80 °C overnight, freeze-dried, sputter-coated with gold, and the micro-morphology was observed by SEM.

[0064] 4. Mechanical property characterization of the BNC integrated film-coated stent

[0065] The bare metal stent and the BNC film-coated stent were respectively compressed and released with tweezers to evaluate the compressibility of the stent. Further, the bare metal stent and the BNC film-coated stent were respectively radially compressed by 2 mm, 4 mm, 6 mm and 7 mm using a universal testing machine, and each distance was cyclically compressed 10 times. The change of the compression force with time was recorded to evaluate the anti-fatigue performance of the stent and the stability of the film coating. The bare metal stent and the BNC film-coated stent were implanted into the trachea of New Zealand white rabbits, and the shrinkage and release process of the stent was monitored by DSA to evaluate whether the mechanical properties of the BNC film-coated stent could meet the requirements of interventional delivery surgery.

[0066] 5. Preparation of the PTX-loaded film-coated stent and PTX release curve

[0067] PTX was dissolved in an aqueous solution of 1% (v / v) ethanol. The freeze-dried BNC film-coated stent was incubated in 0.25 mg / ml, 0.5 mg / ml, 1 mg / ml, 2 mg / ml and 4 mg / ml for 3 h and then taken out. Take about 2 mm 2The PTX-loaded BNC film was immersed in a solution containing 10 wt% Tween-80 and shaken overnight. Then, the supernatant was taken to measure the PTX content and calculate the total amount. At 2 h, 8 h, 24 h, and 48 h, 1 ml of the supernatant was taken to measure the PTX content, and the release curve was obtained.

[0068] 6. Cytotoxicity and subcutaneous embedding toxicity detection of BNC and BNC-PTX drug-loaded integrated covered stents

[0069] (1) CCK-8 analysis of cytotoxicity

[0070] The BNC and BNC-PTX covered stents were taken, immersed in 10 mL of complete DMEM medium after 24 h of ultraviolet radiation, and the leachate was taken on the 1st, 3rd, and 5th days, filtered through a 0.22 μm filter membrane, and co-incubated with human bronchial epithelial cells HBE16 respectively. After 2, 4, 8, 12, and 24 h, the CCK-8 kit was used to evaluate cytotoxicity.

[0071] (2) Confocal observation of cell morphology on the surface of BNC film

[0072] After the purified BNC film was autoclaved, it was incubated in a solution containing complete medium for 1 hour. The prepared BNC was laid in a 24-well plate, and 16HBE was seeded at a density of 1×10 5 in the 24-well plate. It was incubated in an incubator at 37 °C and 5% CO2. After 12 h, 24 h, and 48 h respectively, the supernatant was removed, fixed with 4 wt% paraformaldehyde for 4 h, permeabilized with 0.1 wt% Triton for 5 min, incubated with 1 wt% BSA for 30 min to remove specific adsorption, and DAPI and phalloidin were incubated successively for 30 min. Between each step, it was washed 3 times with PBS. After staining, the cell morphology was observed and recorded with a confocal microscope.

[0073] (3) Subcutaneous embedding toxicology detection in rats.

[0074] Six adult male rats were used. After local hair removal on the left hind limb, a BNC film piece with a size of 0.3 cm was subcutaneously embedded. Then, it was observed for 4 weeks, and the diet, body weight, and local changes were recorded. After sacrifice, the local tissue was stained with HE.

[0075] (4) Establishment of a tumor-bearing mouse model and subcutaneous embedding detection.

[0076] Nude mice BALB / c were divided into an experimental group and a sham operation group, with 6 mice in each group. The lung adenocarcinoma cell line A549 was subcutaneously implanted in the left lower limb. The tumor diameter was about 5 mm. A BNC film square with a diameter of about 0.5 mm was implanted on the surface of the subcutaneous tumor. In the sham operation group, only the epidermis was incised. It was observed for 4 weeks, and the body weight change was recorded. After sacrifice, the tumor size was recorded and stained with HE.

[0077] 7. In vivo tracheal implantation of BNC integrated covered stents

[0078] Nine adult New Zealand rabbits were used and divided into 3 groups: bare stent group, BNC membrane group, and PTX-loaded BNC membrane group, with 3 rabbits in each group. The rabbits were anesthetized by intravenous injection of pentobarbital into the marginal ear vein. Then, a stent was implanted at the tracheal position under DSA. The animals were euthanized at the first week and the second week respectively. Tissues from the proximal, central, and distal ends of the stent were taken, fixed with 4wt% paraformaldehyde, embedded in paraffin, sectioned, and stained with HE, PAS, MASSION, and immunofluorescence (β-tubulin, CD45). The intensity of immunofluorescence positive signals was evaluated by Image Pro Plus software (Media Cy.rnetics, Silver Spring, MD, USA).

[0079] 8. Construction of customized asymmetric BNC-covered stent

[0080] (1) Mold construction

[0081] Clinical tracheal data (0.625mm, 256*256) were obtained using a 64-detector row CT (simens, USA), and the STL format file was exported after processing and annotation on the workstation. A hollow model was made using CAD and the mold was printed using a PLC machine (novapo, China).

[0082] (2) Preparation of asymmetric drug-loaded membrane. The asymmetric BNC-covered membrane was prepared using the mold by the aforementioned method and co-incubated with FITC-PTX overnight.

[0083] 9. Statistical analysis

[0084] The statistical significance of the cytotoxicity experiment between the treatment group and the negative control group was calculated by one-way analysis of variance (ANOVA), and a P value < 0.001 was considered significant. SPSS statistical analysis was used (version 27.0; IBM Corp., Armonk, New York, USA).

[0085] II. Experimental results and analysis

[0086] 1. Metal stent coated with CaO2 nanoparticles

[0087] The macroscopic and microscopic structures of two metal stents (GelMA and CaO2-GelMA) coated with hydrogel and hydrogel loaded with CaO2 nanoparticles are shown in A and B below. From the macroscopic structure, it can be seen that there is a layer of hydrogel coating on the surface of both metal stents. The elemental distribution and semi-quantitative analysis of the two metal stents by Mapping scanning are shown in Figure 2 as follows. The results are shown in Figure 2As shown in C, it can be seen that there is no obvious Ca distribution on the surface of the GelMA scaffold, while obvious and uniform Ca element distribution can be seen at the positions coated with hydrogel on the CaO2-GelMA scaffold, accounting for about 1% of the total elements, which can prove the successful loading of CaO2 nanoparticles. Further, when the two metal scaffolds are placed in water, the results are as Figure 9 shown in A. On the surface of the GelMA scaffold, no bubbles are generated, while on the surface of the CaO2-GelMA scaffold, obvious and fine bubbles can be seen, and these bubbles can be generated continuously for several days, which can prove that the CaO2-GelMA scaffold can continuously generate oxygen when placed in a liquid.

[0088] 2. BNC integrated film-covered stent

[0089] After inoculating the fermentation broth into the reactor and culturing for 3 days, the macroscopic morphology of the film-covered stent was observed, and live / dead bacteria staining was carried out. The results are as Figure 3 shown in A. It can be seen from the cross-section of the reactor that the hydrogel-like BNC has filled the entire lumen and evenly wrapped the metal stent. From the live / dead bacteria staining, it can be seen that the metal stent is surrounded by bacteria driven by oxygen. Further, after taking out and purifying the film-covered stent cultured for 14 days from the reactor, SEM observation was carried out. The results are as Figure 3 shown in B. A BNC film with a nanofiber diameter is coated on the surface of the metal stent. The element distribution and element content of the bare metal stent and the film-covered stent are as Figure 3 shown in C and D. From the surface analysis, it can be known that the ratios of Ti, Ni, C, and O on the surface of the bare metal stent are 17.9%, 21.98%, 40.47%, and 18.61% respectively, while the ratios of Ti, Ni, C, and O on the surface of the film-covered stent are 4.1%, 4.75%, 51.01%, and 39.24% respectively. The element distribution difference is caused by the BNC film on the surface. From SEM and element distribution, we have successfully constructed an integrated BNC film-covered stent.

[0090] The present invention uses a nitinol stent (with a diameter of 8 mm) and constructs an integrated film-covered stent by wrapping the metal stent with BNC, which is different from the traditional hot pressing method and sewing method, enhances the connection between the film and the metal frame, shows the growth of bacteria and fiber winding on both the inner and outer surfaces of the metal stent, and the film is dense and continuous under SEM.

[0091] 3. Repeated compression performance of BNC integrated film-covered stent

[0092] As an important material in biomedical applications, the mechanical properties of BNC film imitating human tissues are widely used to study cell behavior and tissue repair. In the present invention, BNC shows a high tensile strength (1.2 MPa), as Figure 6As shown in A. Further, the naked stent and the integrated BNC covered stent were respectively compressed and released using forceps, and the results are as Figure 6 shown in B. Both the naked stent and the BNC covered stent can be repeatedly compressed and released. Further, the naked stent and the BNC covered stent were respectively radially compressed by 2 mm, 4 mm, 6 mm, and 7 mm using a universal tester, and each distance was cyclically compressed 10 times. The compression procedures of the two stents were the same, and the change process of the compression distance with time is as Figure 6 shown in C. The fitting curves of the compression force and the compression distance are as Figure 6 shown in D. For the change of the compression force with time, the forces of different cycles were statistically analyzed separately, as Figure 6 shown in E. It can be seen that at the same compression distance, there is no significant difference between the compression force of the BNC covered stent and that of the naked stent, indicating that the BNC covering does not significantly affect the compression force of the stent, and after repeated compression, there is no significant difference in the compression force, indicating that the BNC covered stent has excellent fatigue resistance, can withstand the compression and release during the interventional surgery implantation process, and after repeated compression, the BNC film will not fall off or tear, and has excellent stability, always maintaining the structure of the integrated covered stent.

[0093] Further, the naked stent and the BNC covered stent were implanted into the trachea of New Zealand white rabbits, and DSA can monitor the dynamic images of the stent implantation process. The results are as Figure 6 shown in F and G. It can be seen that both the naked stent and the BNC covered stent can be shrunk and delivered to the designated position and successfully released, proving that the BNC covered stent can be successfully delivered to the designated area of the trachea, and the shrinking and releasing are not affected by the covering.

[0094] The integrated BNC covered stent prepared by the present invention has high integrated stability. As Figure 10 shown, after being compressed 10,000 times, it still maintains the integrated characteristics of the film and the stent.

[0095] 4. In vitro and in vivo biocompatibility of the BNC integrated covered stent

[0096] The antigenicity of BNC is extremely low, and currently, most studies apply it to cell growth platforms or cell transplantation carriers. In the present invention, 16HBE was seeded on the surface of BNC. After fluorescence confocal microscopy analysis, it can be seen (as Figure 4 shown in A) that the cells survived and proliferated within 48 h. At the same time, CCK8 data supported that BNC has no obvious toxicity to human bronchial epithelial cells 16HBE. As Figure 4 shown in B and C.

[0097] Further, 1 mm 2The material was embedded subcutaneously in rats, showing no obvious toxicity or side effects within the 4-week observation period (see Figure 9 B).

[0098] 5. Evaluation of the in vitro and in vivo killing ability of the PTX-loaded BNC integrated covered stent

[0099] BNC membranes have been widely studied in the field of drug delivery due to their non-toxicity, high liquid absorption rate, and retention ability. As shown by SEM ( Figure 3 B), the nanofibrils of the nanocellulose membrane are evenly entangled, which provides sufficient porosity for the film and helps the sustained release of drugs. In this invention, PTX was further used as a model drug to investigate the drug loading / in vitro and in vivo release behavior of the BNC membrane (as shown in Figure 4 D). The drug loading of the PTX-loaded BNC membrane reached its peak when incubated at 2 mg / ml, as shown in Figure 4 E, and at the same time, the concentration of the supernatant solution was stable at 2 mg / ml within 48 h, as shown in Figure 4 F. Co-culture with A549 showed an obvious killing effect, as shown in Figure 4 G. Further, we constructed a subcutaneous tumor-bearing mouse model. The experiment of embedding with the PTX-loaded BNC membrane showed obvious differences in tumor size. Histological section analysis showed that the growth of the experimental group was restricted, as shown in Figure 9 B-D.

[0100] 6. Preparation of the asymmetric drug-loaded covered stent and in vitro animal experiments

[0101] Based on the clinical annotation data (red indicates the tumor contact part), a three-dimensional model of the contact between central lung cancer and the trachea was successfully reproduced ( Figure 5 i), and the model file was exported. According to the three-dimensional model, a PCL mold with a hollowed-out contact area was printed ( Figure 5 ii), and the printed mold, metal stent, and acrylic mold were assembled into a BNC membrane culture dish to prepare a metal stent asymmetrically covered with BNC ( Figure 5 iii). The final product ( Figure 5 iv) showed that a locally covered BNC membrane was successfully formed in the corresponding area and FITC-PTX was loaded. Under ultraviolet irradiation, the BNC membrane loaded with FITC-PTX turned green ( Figure 5 , iv). Using a porcine trachea similar in caliber to the bronchus to simulate the stent implantation process, it was found that the stent adapted to the deformation during the implantation process, and the locally covered BNC membrane did not shift or fall off ( Figure 5 , v). 72 h after stent implantation, the bronchus was longitudinally dissected to remove the stent. Under ultraviolet light, strong fluorescence accumulation was visible in the area covered by the BNC membrane ( Figure 5 , vi). The above results prove that this invention has successfully achieved customized coverage and drug release according to the clinical data area.

[0102] Meanwhile, it has flexibility to adapt to the anatomical features of patients, avoid stent migration and intimal injury, and reduce granulation tissue reaction. In stent design, the contact area of most stents with the intima is considered for maintaining radial support rather than for biological functions. As a result, it is difficult for the covered normal area to maintain normal physiological functions, especially in the trachea. The main reason that patients cannot tolerate is that the tracheal mucosa affected by the implant secretes a large amount of mucus that cannot be discharged. Clinical data studies support that partially covered self-expanding metal stents have a longer patency rate and a lower migration rate. Currently, most of the partially covered self-expanding metal stents mainly have annular hollowing at the ports. The research of the present invention clearly shows the feasibility of customizable film coating design based on metal stents and shows promising results in simulation experiments.

[0103] 7. In Vivo Tracheal Implantation of BNC Integrated Film-Coated Stent

[0104] (1) Influence of BNC Stent on Mucosa Compared with Bare Stent

[0105] Two weeks after the implantation of the BNC stent, the tracheal mucosa morphology in the film-coated area exists, while obvious tracheal necrosis is observed around PTX-BNC (shown by HE). At the same time, β-tubulin staining shows that the tracheal mucosa survives, while CD45 shows no obvious inflammatory cell infiltration ( Figure 7 A and B in). This study shows that there are increases in CD45 and PAS shows an increase in goblet cells in the short term (1 week) after the implantation of the bare stent, and it recovers around 2 weeks, as Figure 8 shown; suggesting local inflammatory infiltration caused by acute surgery. In the area covered by the BNC membrane, there is still less CD45 infiltration in the tracheal mucosa 2 weeks later, suggesting that the immune cell infiltration caused by BNC is extremely rare and does not trigger a continuous inflammatory infiltration effect.

[0106] (2) In Vivo Effect of PTX-BNC Stent

[0107] In the area covered by the PTX-BNC membrane, there is no obvious β-tubulin staining, while CD45 staining shows intensive aggregation, suggesting local mucosal necrosis and inflammatory cell infiltration. Similar results are shown around the membrane. Figure 7 C in shows the differences in the two immunostainings among the three groups. It is suggested that PTX effectively releases and exerts a killing effect in vivo.

[0108] III. Conclusion

[0109] The present invention utilizes the unique property of Gluconacetobacter xylinus to produce bacterial nanocellulose (BNC) under aerobic conditions. By releasing oxygen through a metal scaffold loaded with CaO2 particles, it drives the colonization of bacteria around the metal scaffold and gradually generates nanocellulose. Eventually, the nanocellulose wraps the metal network 360 degrees to construct an integrated covered stent, and further realizes drug loading and slow release. This integrated covered stent has undergone 100 times of repeated compression, and its mechanical properties have no obvious change compared with the bare stent; and when the covered stent is compressed to 90%, the stress is about 3.5 N, still maintaining a high integration degree. Further in vivo animal experiments show that after implanting into the trachea of New Zealand white rabbits for 2 weeks, the tracheal patency rate is 100%, the tracheal epithelial cells in the covered area survive, and there is no obvious inflammatory cell infiltration or goblet cell metaplasia. Finally, based on 3D printing modeling of clinical data, a customized covering scheme on the metal stent was designed. The pig trachea simulation experiment shows that the customized covered stent loaded with FITC-PTX can achieve precise drug delivery. In summary, this integrated covered stent constructed by in-situ microbial synthesis of nanocellulose has high integration degree, large drug loading capacity, good biocompatibility and in-vivo safety, and is a very promising covered stent platform that can be applied to various in-vivo cavity interventional therapies.

[0110] Comparative Example 1

[0111] Referring to the method of Example 1, the method without using oxygen drive was investigated, that is, the bare metal stent was directly placed in a mold for fermentation to prepare a BNC covered stent, and the covered stent was taken out after the fermentation was completed. It was found that it was easy to produce the phenomenon of separation between the membrane and the stent by using this method, and it was difficult to achieve integrated preparation.

Claims

1. A preparation method of an in-situ synthetic integrated film-covered stent for microorganisms, characterized in that, It includes the following steps: (1) Mix CaO2 nanoparticles with a GelMA hydrogel solution, and then immerse the metal stent into the hydrogel solution; after taking it out, cure it under ultraviolet light to obtain a metal stent loaded with CaO2 nanoparticles; (2) Inoculate Gluconacetobacter xylinus into a fermentation medium, and culture it with shaking to obtain an activated bacterial culture solution; (3) Prepare a reactor by fixing the metal stent loaded with CaO2 nanoparticles between two silicone tubes with different inner and outer diameters. Inoculate the fermentation broth filled with the activated bacteria obtained in step (2) into the sterilized reactor, and place it in a static culture at 30 °C for 14 days to obtain a nanofibrillated cellulose-coated stent. After removing the reactor, purify the nanofibrillated cellulose-coated stent to obtain the integrated coated stent.

2. The method according to claim 1, wherein In step (1), the mass-volume concentration of the GelMA hydrogel solution is 7.5%, and the addition amount of the CaO2 nanoparticles in step (1) is 0.01-1% by mass-volume concentration.

3. The method according to claim 1, wherein In step (1), the metal stent includes any one of a nitinol stent, a cobalt-chromium alloy, a 316L stainless steel stent, a nickel stent, a tantalum stent, a magnesium stent, and a zinc stent.

4. The method according to claim 1, wherein In step (1), the curing is carried out by irradiating with ultraviolet light of 365 nm for 5 min.

5. The method according to claim 1, wherein In step (2), the formula of the fermentation medium is: 100 g / L D-fructose, 5 g / L peptone, and 3 g / L yeast extract, and the pH is 5.

0.

6. The method according to claim 1, wherein The sources of the Gluconacetobacter xylinus described in step (2) include the strains preserved on slants, the strains preserved in cryopreservation solutions, and the bacterial cellulose membranes with strains. The inoculation amount of the strains preserved on slants is 1 - 2 loops, the inoculation amount of the strains preserved in cryopreservation solutions is 1 - 5% (v / v), and the inoculation amount of the bacterial cellulose membranes with strains is 1 - 5 mm 3 of 1 - 5 pieces of bacterial cellulose membranes.

7. The method according to claim 1, characterized in that In step (2), the culture in the fermentation medium is carried out with shaking at 30 °C and 120 rpm, and the static culture is carried out in a constant temperature and humidity incubator at 30 °C for 7 days.

8. The method according to claim 1, wherein In step (3), the inner and outer diameters of the two silicone tubes are adjustable sizes, and the inner and outer diameter sizes of the inner and outer silicone tubes range from 1.5-30 mm * 2-35 mm, and the inner diameter difference between the outer silicone tube and the inner silicone tube is 0.5-10 mm.

9. An integrated coated stent prepared by the method according to any one of claims 1-8.

10. Use of the integrated coated stent according to claim 9 in the preparation of a tissue engineering scaffold material.

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