In-situ synthesis of a microbial integrated stent with a film and a preparation method and application thereof

Abstract

This invention provides an integrated microbial in-situ synthesized coated scaffold, its preparation method, and its application, belonging to the field of tissue engineering scaffold material technology. The invention involves coating a metal scaffold surface with CaO2 nanoparticles using photocurable hydrogel, fixing it in a customized reactor filled with fermentation broth. The slow hydrolysis of the CaO2 nanoparticles continuously releases oxygen, driving *Acetobacter xylinum* to colonize and proliferate on the metal scaffold surface. The bacteria gradually synthesize nanocellulose in situ, ultimately achieving 360-degree encapsulation of the scaffold's metal mesh by the nanocellulose, resulting in an integrated scaffold and coating. The coated scaffold of this invention exhibits structural stability after 10,000 cycles of compression, with the stress of both the bare metal scaffold and the coated scaffold at 90% compression being only 3.5 N, achieving a mechanical stability of 99.999%. Furthermore, this integrated coated scaffold features high-capacity drug loading, high biocompatibility, and high in vivo safety.

Description

Technical Field

[0001] This invention belongs to the field of tissue engineering scaffold material technology, specifically relating to an integrated microbial in-situ synthesized membrane scaffold, its preparation method, and its application. Background Technology

[0002] The treatment strategy of replacing diseased cavities with artificial tubular stents is widely used in cavities such as the cardiovascular system, esophagus, trachea, intestines, bile ducts, and urethra. Most cavities possess superior mechanical properties in their outer sheath support and a biologically functional endothelial system to achieve their respective physiological functions. Currently, apart from drug-eluting small-diameter vascular stents, no other biologically functional stents are commercially available. Among commercial stents, plastic tubular stents are limited by mechanical properties and rejection issues, hindering long-term implantation and the fabrication of large-diameter stents. Silicone stents have high migration rates and high prices, preventing their widespread application. Metal stents possess high deformation strength, fatigue resistance, and low migration rates, making them suitable for minimally invasive placement and removal; their mechanical properties are irreplaceable in maintaining the morphology of cavities. Improving the covering scheme based on metal stents to achieve biological functions is currently the most translational approach.

[0003] Covered scaffolds are becoming increasingly important as an alternative to surgical procedures in modern medicine. However, existing covered scaffolds generally suffer from problems such as easy detachment of the covering, poor compliance, and poor tissue compatibility, which seriously hinder the development of tissue engineering scaffolds.

[0004] Currently, clinically available covered scaffold products based on metallic scaffolds include Cook Medical's Zenith, Medtronic's Talent, Endurant, and AneuRx, Gore's Excluder, and Boston Scientific's Vanguard, among others. The covering materials primarily include polytetrafluoroethylene (PTFE), polyester fiber, and polyester filament. These synthetic materials only provide physical isolation and lack biological function. Research on materials for biofunctional metallic scaffolds focuses on developing novel biomaterials, such as hydrogels and decellularized matrices, primarily for single-tissue regeneration, such as bone. The integration with tubular metallic scaffolds often relies on simple surface attachment. This type of covered scaffold construction suffers from a mismatch in mechanical properties after membrane formation, easily leading to membrane detachment and re-blockage of cavities. Decellularized matrices are costly to prepare and cannot provide precise matching in terms of the size and mechanical properties of specific patient cavities, posing a risk of in vivo collapse. The integration of the membrane with the metallic scaffold determines the long-term safety of the scaffold implantation and is a decisive factor in the clinical translation of covered scaffolds.

[0005] Therefore, existing covered scaffolds generally suffer from problems such as easy detachment of the covering, poor compliance, and poor tissue compatibility, which seriously hinder the development of tissue engineering scaffolds.

[0006] 3D printing can utilize composite materials to construct multilayer tubular scaffolds. However, due to the low toughness of existing materials, there is no complete alternative to metal scaffolds for applications requiring high stress and plastic deformation. Electrospinning has been reported to construct covered scaffolds for skin and urethral repair. However, the selection of electrospinning materials is limited, and inherent defects such as poor mechanical stability of natural materials, poor biocompatibility of synthetic materials, and difficulties in mass production of hybrid materials hinder its application and make additive manufacturing based on metal frameworks difficult. Therefore, there is an urgent need for new methods to construct integrated covered metal scaffolds to achieve biological functions.

[0007] However, there are currently no customizable coating solutions based on metal scaffolds. This may be related to the way other materials are bonded to metal scaffolds, where the surface contact area is too small to generate sufficient adhesion, or the formation process cannot be customized. This can also lead to over-coverage interfering with normal endothelial function and resulting in adverse clinical outcomes.

[0008] Bacterial nanocellulose (BNC), as a novel biomaterial, has been widely studied for its applications in implants and drug delivery systems. It possesses a 3D ultrafine fiber mesh structure, excellent mechanical properties and biocompatibility, and its production process is simple and customizable. Cherng et al. successfully used BNC as a matrix to maintain the stemness of epithelial cells and induce differentiation. Baoxiu Wang et al. constructed a porous scaffold based on bacterial nanofibers and successfully used it for urethral repair. BNC exhibits high tensile strength and excellent flexibility, but lacks support, making it difficult to maintain its tubular morphology without external support. Its tensile strength originates from its unique fiber network structure and strong hydrogen bonding, allowing BNC to be generated into arbitrary shapes while maintaining its surface integrity. Furthermore, thanks to the unique production mode of gradual bacterial community formation, implants in the BNC membrane production environment can be completely encapsulated by the BNC membrane, achieving the same tensile strength. That is, the tensile strength at the material edges does not change drastically due to interfacial variations, leading to breakage; instead, the BNC membrane provides a completely continuous coverage, resulting in uniform tensile strength. Meanwhile, the BNC membrane maintains its microscopic fiber network structure, thus preserving its flexibility. This is significantly different from the hot-pressing and stitching methods used in current coating materials. BNC-metal hybrid materials often employ a BNC framework and different metal fillers to obtain additional electrical and thermal conductivity, but the method of coating BNC with a metal framework has not yet been reported.

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

[0010] The present invention aims to solve the aforementioned technical problems by providing an integrated microbial in-situ synthesized membrane scaffold, its preparation method, and its application. The technical objective of this invention is to address the limitations of existing methods in successfully preparing integrated membrane-coated metal scaffolds, and the difficulty in preparing BNC membrane-coated integrated metal scaffolds with low rejection rates, low production costs, and high customizability.

[0011] To achieve the above-mentioned technical objectives, the technical solution adopted by the present invention is as follows:

[0012] This invention first provides a method for preparing an integrated membrane scaffold synthesized in situ by microorganisms, comprising the following steps:

[0013] (1) CaO2 nanoparticles were mixed with GelMA hydrogel solution, and then the metal scaffold was immersed in the hydrogel solution; after being taken out, it was cured under ultraviolet light to obtain a metal scaffold loaded with CaO2 nanoparticles.

[0014] (2) Inoculate *Acetobacter xylosinophila* into the fermentation medium, shake and culture to obtain the activated bacterial culture solution;

[0015] (3) A reactor is prepared by fixing a metal scaffold loaded with CaO2 nanoparticles between two silicone tubes with different inner and outer diameters. The fermentation broth filled with activated bacteria obtained in step (2) is inoculated into the sterilized reactor and placed at 30°C for static culture for 14 days to obtain a nanocellulose-coated scaffold. After removing the reactor, the nanocellulose-coated scaffold is purified to obtain the integrated coated scaffold.

[0016] This invention utilizes a method to prepare a metal scaffold loaded with CaO2 particles, which slowly releases oxygen to drive bacterial colonization around the scaffold, gradually generating nanocellulose. This achieves 360-degree encapsulation of the metal network by the nanocellulose, thus successfully constructing an integrated membrane-coated scaffold. This invention mixes CaO2 nanoparticles with GelMA hydrogel to construct a CaO2 nanoparticle-coated metal scaffold, which releases oxygen, thereby driving bacterial colonization around the scaffold and achieving an integrated BNC membrane-coated scaffold. Compared to later physical integration of other membranes with the scaffold, this integrated approach avoids membrane-scaffold separation.

[0017] On the other hand, in this invention, the small amount of GelMA hydrogel and CaO2 nanoparticles used in the coating process only serve to release oxygen. As the fermentation process proceeds, the above materials will be completely degraded within 5 days. Therefore, the final product is a pure BNC-coated scaffold that does not contain GelMA hydrogel and CaO2 nanoparticles. This is fundamentally different from composite materials of GelMA hydrogel or other nanoparticles and bacterial cellulose.

[0018] The integrated BNC-coated scaffold prepared by the method of this invention has high integration stability, and still maintains the integration of membrane and scaffold after 10,000 compressions. However, when trying to use a method that does not utilize oxygen, i.e., directly placing the metal scaffold in a mold for fermentation to prepare the BNC-coated scaffold, the membrane and scaffold easily separate after fermentation, making it difficult to achieve integration.

[0019] This invention demonstrates that, in vitro, the coated scaffold exhibits excellent cell compatibility, enabling effective drug loading and slow release. Simultaneously, in vivo, it allows for targeted drug delivery with minimal damage and inflammation to the airway mucosa. Furthermore, this invention utilizes a 3D-printed template to achieve an asymmetric coated drug-loaded design on a nickel-titanium alloy tracheal stent. This design retains the support and deformation capabilities of the metal stent while effectively defining the coating area, enabling customized coating within a fixed range and precise drug release within the defined region.

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

[0021] (1) Existing coated stents cannot be used to prepare integrated coated stents. The membrane is prone to detachment and separation from the metal stent. However, the method of the present invention can prepare integrated coated stents without membrane separation and detachment.

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

[0023] (3) Existing methods directly prepare membrane-coated scaffolds through microbial fermentation. As shown in the comparative example, this method results in the separation of the scaffold from the membrane. However, the method of this invention, combined with an oxygen-driven method, successfully prepares an integrated membrane-coated scaffold, thus achieving the integrated preparation of the membrane-coated scaffold.

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

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

[0026] Furthermore, the curing process described in step (1) involves irradiation with 365nm light for 5 minutes.

[0027] Furthermore, the fermentation medium in step (2) is formulated as follows: 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 *Acetobacter xylinum* mentioned in step (2) include strains preserved on slant agar, strains preserved in cryopreservation solution, and bacterial cellulose membranes containing strains. The inoculum size for strains preserved on slant agar is 1-2 loops, the inoculum size for strains preserved in cryopreservation solution is 1-5% (v / v), and the inoculum size for bacterial cellulose membranes containing strains is 1-5 mm. 3 1-5 pieces of bacterial cellulose membrane.

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

[0030] Furthermore, the inner and outer diameters of the two silicone tubes mentioned in step (3) are adjustable, with the inner and outer diameters ranging from 1.5-30mm*2-35mm, and the difference between the inner and outer diameters of the outer and inner silicone tubes being 0.5-10mm.

[0031] The second objective of this invention is to provide an integrated film-coated scaffold prepared by the method described above.

[0032] A third objective of this invention is to provide the application of the integrated covered scaffold described above in the preparation of tissue engineering scaffold materials.

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

[0034] This invention involves coating a metal scaffold with CaO2 nanoparticles using a photocurable hydrogel, which is then fixed in a custom-designed reactor filled with fermentation broth. The slow hydrolysis of the CaO2 nanoparticles continuously releases oxygen, driving *Acetobacter xylinum* to colonize and proliferate on the metal scaffold surface. The bacteria gradually synthesize nanocellulose in situ, ultimately achieving a 360-degree encapsulation of the scaffold's metal mesh, resulting in an integrated scaffold with a coating. Live and dead bacteria staining of the coated scaffold during synthesis confirmed that the bacteria colonized the metal scaffold in situ under oxygen-driven conditions. After 10,000 cycles of compression, the scaffold structure remained stable, with a stress of 3.5 N for both the bare metal scaffold and the coated scaffold at 90% compression, demonstrating a mechanical stability of 99.999%. Two weeks after implantation of the coated scaffold into the trachea of ​​New Zealand white rabbits, tracheal patency was 100%, and 100% of the tracheal epithelial cells in the coated area survived, with no inflammatory cell infiltration or goblet cell metaplasia. Using 3D printing modeling based on clinical data, a pig trachea simulation experiment demonstrated that a customized covered scaffold loaded with FITC-PTX can achieve precise drug delivery. This integrated covered scaffold, constructed from microbially synthesized nanocellulose, features high-capacity drug loading, high biocompatibility, and high in vivo safety, making it a highly promising covered scaffold platform applicable to various intracavitary interventional therapies. Attached Figure Description

[0035] Figure 1 This is a schematic diagram illustrating the fabrication of the integrated film-coated scaffold of the present invention.

[0036] Figure 2 Preparation and physicochemical characterization of scaffolds loaded with CaO2 nanoparticles; A: Schematic diagram of the preparation process; B: Macroscopic morphology and microscopic morphology observed by SEM of metal scaffolds coated with GelMA and GelMA loaded with CaO2 nanoparticles, respectively; C: Elemental distribution maps (Ca / Ti / Ni / C / O / Merge) and elemental content statistics of GelMA and GelMA loaded with CaO2 nanoparticles through mapping.

[0037] Figure 3 Preparation and physicochemical characterization of bare metal scaffolds and BNC-coated scaffolds; A: Macroscopic morphology of BNC-coated scaffolds during in-situ microbial synthesis and distribution of live and dead bacteria observed using fluorescence microscopy; B: Microscopic morphology of purified BNC-coated scaffolds; C: Elemental distribution maps (Ti / Ni / C / O / Merge) of bare metal scaffolds and BNC-coated scaffolds via mapping; D: Elemental content statistics.

[0038] Figure 4For cell compatibility evaluation: A: Schematic diagram of cell compatibility evaluation using extract and co-culture; B: Cytotoxicity test of extract from BNC-coated scaffolds at 1, 3, and 7 days; C: Cytoskeleton fluorescence staining results (F-actin / DAPI) after co-culturing on the BNC membrane surface for 12, 24, and 48 hours; D: Schematic diagram of cell compatibility test of cell extract from BNC-coated scaffolds loaded with PTX; E: Total PTX release from BNC membranes incubated with different PTX concentration solutions; F: PTX release curves with different drug loadings; G: Cytotoxicity of scaffolds with different PTX drug loadings.

[0039] Figure 5 The preparation process and application of asymmetric covered scaffolds.

[0040] Figure 6 Evaluation of the mechanical properties of BNC-coated stents: A: Stress-strain curves of pure BNC membranes; B: Macroscopic compression-release diagrams of bare metal stents and BNC-coated stents; C: Curves showing the changes in stress over time for bare metal stents and BNC-coated stents at compression distances of 2, 4, 6, and 7 mm, respectively; D: Curves showing the changes in stress over compression distance for bare metal stents and BNC-coated stents at compression distances of 2, 4, 6, and 7 mm, respectively; E: Statistical analysis of the average stress at each compression distance for bare metal stents and BNC-coated stents at 2, 4, 6, and 7 mm; FG: DSA observation of stent compression and release when bare metal stents and BNC-coated stents are placed in the rabbit trachea.

[0041] Figure 7 The results are as follows: A) HE / PAS / MASSION staining, and β-tubulin and CD45 fluorescence staining in rabbits 2 weeks after implantation of bare metal scaffolds and drug-loaded / non-drug-loaded covered scaffolds; B) Differences in two immunostaining methods among the three groups with different treatments; C) Statistical graph of β-tubulin / CD45 fluorescence signal intensity in bare metal scaffolds and drug-loaded / non-drug-loaded fully covered scaffolds.

[0042] Figure 8 HE / PAS / MASSION staining, as well as β-tubulin and CD45 fluorescence staining, were performed on rabbits 1 day, 1 week, and 2 weeks after implantation of bare metal scaffolds and drug-loaded / unloaded covered scaffolds. Model scaffold indentations were visible at the scaffold-mucosal contact surface. PAS staining showed an increase in goblet cell model, but less CD45 staining on the surface. β-tubulin staining showed that the mucosa remained viable throughout.

[0043] Figure 9 This is a performance test of the integrated film-coated stent of the present invention.

[0044] Figure 10 These are images of the film-coated stent of the present invention before compression (A) and after 10,000 compressions (B). Detailed Implementation

[0045] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is described in detail below with reference to embodiments. It should be noted that the following embodiments are for explanation and illustration only and are not intended to limit the invention. Non-essential improvements and adjustments made by those skilled in the art based on the above description are still within the scope of protection of this 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 were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Tryptone, yeast extract, and D-fructose were purchased from Adamas by Shanghai Titan Technology Co., Ltd. (Shanghai, China). The *Komagataeibacter xylinus* (K. xylinus) used to produce nanocellulose (BNC) was obtained from the laboratory of Donghua University, with the China Microbial Integrated Culture Collection number 1186. Human bronchial epithelial cells (HBE16) were purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). Fetal bovine serum (FBS), DMEM high-glucose medium, double antibodies (10000 U / mL penicillin, 10000 μg / mL streptomycin), and 0.25% Trypsin-EDTA (1×) were all purchased from Gibco, Shanghai Titan Technology Co., Ltd. (Shanghai, China). Cell counting kit-8 (CCK-8) was purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). Male New Zealand white rabbits (approximately 4 kg) were purchased from SLAC Laboratory Animal Co., Ltd. (Shanghai, China). Nickel-titanium alloy metal scaffolds with outer diameters of 8 mm and 12 mm were from Nanjing MicroPort Medical Technology Co., Ltd. Live / dead bacteria staining kit (SYTO / PI) was purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.

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

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

[0052] The synthesis method of GelMA hydrogel is based on existing literature. 10g of gelatin is added to 100mL of PBS and heated to 60℃ until dissolved and clear. 4mL of MA (methacrylic acid) is added using a micropump at a rate of 250uL / min. The mixture is stirred in the dark and reacted at 60℃ for 2 hours. 100mL of PBS is added to terminate the reaction. The precipitate is removed by centrifugation at 7000rpm for 15min. The gel is dialyzed at 37℃ for 3-4 days using a dialysis bag with a capacity of 8000-14000Da. The gel is then lyophilized to obtain a lyophilized GelMA sample for later use.

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

[0054] 4 mL of 7.5% (m / v) GelMA hydrogel was prepared. 0.04 g of CaO2 nanoparticles were uniformly resuspended in the hydrogel. Metal scaffolds were immersed in both pure GelMA hydrogel and GelMA hydrogel containing CaO2 nanoparticles, respectively, and then cured by irradiation with 365 nm UV light for 5 min. This yielded a metal scaffold encapsulating pure hydrogel (denoted as Gel-Stent) and a metal scaffold loaded with CaO2 nanoparticles (denoted as CaO2-Gel-Stent). The CaO2-Gel-Stent was placed in water, and the generation of bubbles, representing oxygen production, was observed. After freeze-drying, the microstructure was observed using SEM, and elemental distribution and semi-quantitative elemental content were determined by mapping.

[0055] 3. Construction of BNC integrated film-coated scaffold (see...) Figure 1 )

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

[0057] Fermentation medium was prepared using 100 g / L D-fructose, 5 g / L peptone, and 3 g / L yeast extract. The pH was adjusted to approximately 5.0. The medium was autoclaved at 115°C for 30 minutes. After standing at room temperature, *Acetobacter xyloside* colonies were inoculated into the fermentation medium and cultured at 30°C with shaking at 120 rpm until fine flocculent matter was visible to the naked eye, yielding a fermentation broth filled with activated bacteria. The activated bacteria fermentation broth was inoculated into 24-well plates (1 mL per well) and incubated at 30°C for 7 days to obtain BNC membranes.

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

[0059] The metal scaffold loaded with CaO2 nanoparticles was fixed between two silicone tubes with inner diameters of 9*10mm and outer diameters of 6*7mm. Both ends were sealed with silicone plugs. After high-temperature and high-pressure sterilization at 121℃ for 20min, it was placed in a clean bench for later use.

[0060] (3) Culture and observation of bacterial distribution on integrated membrane scaffold

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

[0062] (4) Purification and morphology observation of integrated membrane-coated scaffold

[0063] After 14 days of static culture in a 30℃ constant temperature and humidity incubator, the reactor was removed, and excess culture medium was rinsed with deionized water. The culture system was then placed in a 1% (m / v) NaOH solution and boiled at 80℃ for 6 hours, with the alkali solution replaced every two hours. Subsequently, it was treated with ultrapure water at 80℃ for 48 hours, with repeated replacement of the ultrapure water until the pH reached neutral. Finally, it was placed in a large volume of ultrapure water and treated at 121℃ for 20 minutes five times, with each treatment involving complete replacement of the ultrapure water to remove endotoxins, resulting in a purified BNC-coated scaffold with endotoxin levels meeting the Class III medical device implantation standards. The BNC membrane was purified using the same method. The purified BNC-coated scaffold was immersed in ultrapure water and incubated overnight at -80℃, then lyophilized, sputter-coated with gold, and its microstructure was observed using SEM.

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

[0065] Bare-metal stents and BNC-covered stents were compressed and released using forceps to evaluate their compressibility. Furthermore, a universal testing instrument was used to perform radial compression of the bare-metal stent and BNC-covered stent at 2 mm, 4 mm, 6 mm, and 7 mm, with each compression cycle repeated 10 times. The change in compressive force over time was recorded to evaluate the fatigue resistance of the stent and the stability of the covering. The bare-metal stent and BNC-covered stent were implanted into the trachea of ​​New Zealand white rabbits. DSA was used to monitor the stent's contraction and release process to assess whether the mechanical properties of the BNC-covered stent met the requirements of interventional delivery procedures.

[0066] 5. Preparation of PTX-loaded coated scaffolds and PTX release curves

[0067] PTX was dissolved in a 1% (v / v) ethanol aqueous solution. Lyophilized BNC-coated scaffolds were incubated at concentrations of 0.25 mg / ml, 0.5 mg / ml, 1 mg / ml, 2 mg / ml, and 4 mg / ml for 3 hours, then removed. Approximately 2 mm of the solution was taken. 2A BNC membrane loaded with PTX was immersed in a solution containing 10 wt% Tween-80 and shaken overnight. The supernatant was then collected to determine the PTX content and calculate the total amount. 1 ml of the supernatant was collected at 2 h, 8 h, 24 h, and 48 h to determine the PTX content and obtain the release curve.

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

[0069] (1) CCK-8 assay for cytotoxicity

[0070] BNC and BNC-PTX coated scaffolds were subjected to UV irradiation for 24 hours, followed by soaking in 10 mL of DMEM complete medium. The extracts were collected at days 1, 3, and 5, filtered through a 0.22 μm filter, and co-incubated with human bronchial epithelial cells (HBE16). Cytotoxicity was assessed using a CCK-8 assay kit at 2, 4, 8, 12, and 24 hours.

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

[0072] After sterilizing the purified BNC membranes by autoclaving, they were incubated in a solution containing complete culture medium for 1 hour. The prepared BNC membranes were then plated in 24-well plates, and 16HBE was applied at a rate of 1×10⁻⁶. 5 Cells were seeded in 24-well plates. They were incubated at 37°C with 5% CO2. After 12 h, 24 h, and 48 h, the supernatant was removed. Cells were fixed with 4 wt% paraformaldehyde for 4 h, permeabilized with 0.1 wt% Triton for 5 min, and incubated with 1 wt% BSA for 30 min to remove specifically adsorbed DAPI and phalloidin. Cells were washed three times with PBS between each step. After staining, cell morphology was observed and recorded using a confocal microscope.

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

[0074] Six adult male rats were used. After shaving the hair on the left hind limb, a 0.3 cm BNC membrane was subcutaneously embedded. The rats were observed for 4 weeks, and their diet, weight, and local changes were recorded. After sacrifice, local tissue was collected for HE staining.

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

[0076] Nude BALB / c mice were divided into an experimental group and a sham-operated group, with six mice in each group. Lung adenocarcinoma cell line A549 was subcutaneously implanted into the left hind limb. The tumor diameter was approximately 5 mm. A BNC membrane square with a diameter of approximately 0.5 mm was implanted subcutaneously onto the surface of the tumor. In the sham-operated group, only the epidermis was incised. The mice were observed for 4 weeks, and changes in body weight were recorded. After sacrifice, tumor size and HE staining were recorded.

[0077] 7. BNC Integrated Covered Stent Living Tracheal Implantation

[0078] Nine adult New Zealand rabbits were used and divided into three groups: bare-metal stent group, BNC membrane group, and PTX-loaded BNC membrane group, with three rabbits in each group. Anesthesia was administered via pentobarbital intravenous injection in the ear. The stent was then implanted into the trachea under DSA guidance. Animals were euthanized at the first and second weeks. Tissue samples from the proximal, central, and distal ends of the stent were harvested, fixed in 4wt% paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE), phospholipids (PAS), massion stain, and immunofluorescence stain (β-tubulin, CD45). The intensity of the immunofluorescence signal was assessed using Image Pro Plus software (Media Cyclinics, Silver Springs, MD, USA).

[0079] 8. Construction of customized asymmetric BNC covered scaffolds

[0080] (1) Mold construction

[0081] Clinical tracheal data (0.625mm, 256*256) was obtained using a 64-slice CT scanner (Siemens, USA). The data was processed and annotated on a workstation before being exported as an STL file. A hollow model was created using CAD, and a mold was printed using a PLC printer (Novapo, China).

[0082] (2) Preparation of asymmetric drug-loaded membranes. Asymmetric BNC coatings were prepared using the aforementioned method with a mold and incubated overnight with FITC-PTX.

[0083] 9. Statistical Analysis

[0084] Statistical significance of cytotoxicity assays between the treatment group and the negative control group was calculated using one-way ANOVA, with a p-value <0.001 considered significant. SPSS statistical analysis was performed (version 27.0; IBM Corp. Armonk, New York, USA).

[0085] II. Experimental Results and Analysis

[0086] 1. Metal scaffold coated with CaO2 nanoparticles

[0087] Macroscopic and microscopic structures of hydrogels and hydrogels loaded with CaO2 nanoparticles coated with two metal scaffolds (GelMA and CaO2-GelMA) are shown below. Figure 2 As shown in Figures A and B, the macroscopic structure reveals that both metal scaffolds are coated with a layer of hydrogel. Mapping scans and semi-quantitative analysis of the elemental distribution of the two metal scaffolds yielded the following results: Figure 2As shown in Figure C, it can be seen that no obvious Ca distribution is observed on the surface of the GelMA scaffold, while a clear and uniform distribution of Ca elements, accounting for approximately 1% of the total elements, is visible at the hydrogel-coated areas of the CaO2-GelMA scaffold, demonstrating the successful loading of CaO2 nanoparticles. Furthermore, placing both metal scaffolds in water yielded the following results: Figure 9 As shown in Figure A, no bubbles were observed to be generated on the surface of the GelMA scaffold, while obvious fine bubbles were observed to be generated on the surface of the CaO2-GelMA scaffold, and these bubbles could continue to be generated for several days. This proves that the CaO2-GelMA scaffold can continuously generate oxygen when placed in a liquid.

[0088] 2. BNC integrated film-coated support

[0089] After inoculating the fermentation broth into the reactor and culturing for 3 days, the macroscopic morphology of the membrane-covered scaffold was observed, and live and dead bacteria were stained. The results are as follows: Figure 3 As shown in Figure A, the cross-section of the reactor reveals that the hydrogel-like BNC has completely filled the lumen, uniformly encapsulating the metal scaffold. Staining with live and dead bacteria shows that, driven by oxygen, the metal scaffold is surrounded by bacteria. Furthermore, after 14 days of culture, the coated scaffold was removed from the reactor, purified, and observed using SEM. The results are as follows... Figure 3 As shown in Figure B, a layer of BNC film with a fiber diameter at the nanometer level is coated on the surface of the metal scaffold. The elemental distribution and elemental content of the bare metal scaffold and the coated scaffold are shown in Figure B. Figure 3 As shown in C and D, surface analysis reveals that the proportions of Ti, Ni, C, and O on the surface of the bare metal scaffold are 17.9%, 21.98%, 40.47%, and 18.61%, respectively, while those on the surface of the coated scaffold are 4.1%, 4.75%, 51.01%, and 39.24%, respectively. This difference in elemental distribution is due to the BNC film on the surface. SEM images and elemental distribution data confirm that we have successfully constructed an integrated BNC coated scaffold.

[0090] This invention utilizes a nickel-titanium alloy scaffold (8mm in diameter) and employs a BNC-wrapped metal scaffold to construct an integrated coated scaffold. This method differs from traditional hot-pressing and stitching methods, enhancing the connection between the coating and the metal frame. Microbial growth and fiber entanglement are simultaneously observed on the inner and outer surfaces of the metal scaffold, and the film appears dense and continuous under SEM.

[0091] 3. BNC integrated film-coated stent's repeated compression performance

[0092] BNC membranes, as important materials in biomedical applications, are widely used in research on cell behavior and tissue repair due to their ability to mimic the mechanical properties of human tissues. In this invention, BNC exhibits high tensile strength (1.2 MPa), such as... Figure 6As shown in Figure A. Further, the bare metal stent and the integrated BNC covered stent were compressed and released using tweezers, with the results as follows. Figure 6 As shown in Figure B, both the bare metal stent and the BNC-coated stent can be repeatedly compressed and released. Furthermore, a universal testing instrument was used to perform radial compression of 2mm, 4mm, 6mm, and 7mm on the bare metal stent and the BNC-coated stent, respectively. Each distance was cyclically compressed 10 times. The compression program was the same for both types of stents, and the change in compression distance over time was as follows. Figure 6 As shown in Figure C, the fitting curves for compressive force and compression distance are as follows: Figure 6 As shown in Figure D, the change of compressive force over time is statistically analyzed separately for different cycles, such as... Figure 6 As shown in Figure E, it can be seen that at the same compression distance, there is no significant difference in the compressive force of the BNC-covered stent compared to the bare metal stent. This indicates that the BNC covering does not significantly affect the compressive force of the stent. Furthermore, after repeated compression, there is no significant difference in compressive force, indicating that the BNC-covered stent has excellent fatigue resistance and can withstand the compression and release during interventional surgery. Moreover, after repeated compression, the BNC membrane will not detach or tear, demonstrating excellent stability and maintaining the structure of the integrated covered stent.

[0093] Furthermore, bare-metal stents and BNC-covered stents were implanted into the trachea of ​​New Zealand white rabbits. DSA was used to monitor the dynamic process of stent implantation, and the results were as follows: Figure 6 As shown in F and G, it can be seen that both the bare metal stent and the BNC covered stent can be successfully delivered to the designated location and then released, proving that the BNC covered stent can be successfully delivered to the designated area of ​​the trachea, and that the contraction and release are not affected by the covering.

[0094] The integrated BNC-coated scaffold prepared by this invention has high integration stability, such as... Figure 10 As shown, even after being compressed 10,000 times, it still maintains the property of being integrated with the scaffold.

[0095] 4. In vitro and in vivo compatibility of BNC integrated covered scaffold

[0096] BNCs have extremely low antigenicity, and current research primarily focuses on their application in cell growth platforms or cell transplantation vectors. In this invention, 16HBE was seeded on the surface of BNCs, and fluorescence confocal microscopy analysis revealed (e.g.) Figure 4 In study A), cells survived and proliferated within 48 hours. Furthermore, CCK8 data supported the finding that BNC had no significant toxicity to human bronchial epithelial cells 16HBE. Figure 4 As shown in B and C.

[0097] Furthermore, 1mm 2The material was embedded subcutaneously in rats, and no significant toxicity or side effects were observed within a 4-week observation period (see [link to study]). Figure 9 (B)

[0098] 5. In vivo and in vitro lethality assessment of PTX-BNC integrated covered scaffold.

[0099] BNC membranes have been widely studied in the field of drug delivery due to their non-toxicity, high liquid absorption rate, and retention capacity. For example, SEM ( Figure 3 As shown in Figure B), the nanocellulose membrane fibers are uniformly entangled, which provides sufficient porosity for the film and facilitates sustained drug release. This invention further investigated the drug loading / in vitro / in vivo release behavior of the BNC membrane using PTX as a model drug (e.g., Figure 4 (D). The drug loading of PTX-loaded BNC membranes reached its peak at an incubation time of 2 mg / ml, such as... Figure 4 As shown in Figure E, the supernatant concentration remained stable at 2 mg / ml over a 48-hour period. Figure 4 As shown in Figure F. Co-culturing with A549 showed a significant killing effect, such as... Figure 4 As shown in Figure G. Furthermore, we constructed a subcutaneous mouse model of tumor bearing tumors. The BNC membrane embedding experiment with PTX showed a significant difference in tumor size. Histological analysis revealed that growth was restricted in the experimental group, as shown in Figure G. Figure 9 Chinese Blu-ray.

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

[0101] Based on clinical annotation data (red indicates the tumor contact area), a three-dimensional model of the central lung cancer contacting the trachea was successfully reproduced. Figure 5 (i) and export the model file. Print the PCL mold with cutouts in the contact area based on the 3D model (i). Figure 5 (ii) The printing mold, metal scaffold, and acrylic mold are assembled into a BNC membrane culture dish to prepare a metal scaffold asymmetrically covered with BNC. Figure 5 (iii) Final product ( Figure 5 (iv) As can be seen, a locally covered BNC film was successfully formed in the corresponding area, and FITC-PTX was loaded. Under ultraviolet irradiation, the BNC film loaded with FITC-PTX turned green ( Figure 5 (iv) A porcine trachea with a diameter similar to that of a bronchus was used to simulate the stent implantation process. It was observed that the stent adapted to the deformation during implantation, and the locally covering BNC membrane did not shift or detach. Figure 5 , v). 72 hours after stent implantation, the bronchus was longitudinally perforated and the stent removed. Under UV light, fluorescence concentration was observed in the area covered by the BNC membrane (v). Figure 5 (vi). The above results demonstrate that the present invention successfully achieves customized coverage and drug delivery based on clinical data regions.

[0102] Simultaneously, it possesses flexibility to adapt to patient anatomical features, avoiding stent migration and intimal damage, and reducing granulation tissue reaction. In stent design, the contact area between the stent and the intimal is often prioritized for maintaining radial support rather than for biological function. This makes it difficult for the covered normal area to maintain normal physiological function, especially in the trachea. The inability to drain large amounts of mucus secreted by the implanted tracheal mucosa is a major reason for patient intolerance. Clinical data studies support that partially covered self-expanding metallic stents have longer patency rates and lower migration rates. Currently, partially covered self-expanding metallic stents mainly have annular perforations at the port. The research of this invention clearly demonstrates the feasibility of customizable endothelial design based on metallic stents and shows promising results in simulation experiments.

[0103] 7. BNC Integrated Covered Stent Living Tracheal Implantation

[0104] (1) Effects of BNC stent on mucosa compared to bare stent

[0105] Two weeks after BNC stent implantation, the tracheal mucosa morphology in the covered area remained intact, while significant tracheal necrosis was observed around PTX-BNC (HE staining). Simultaneously, β-tubulin staining showed tracheal mucosal viability, while CD45 staining showed no significant inflammatory cell infiltration. Figure 7 (A and B). This study shows that CD45 levels are elevated and PAS (palatable scaffold) shows an increase in goblet cells in the short term (1 week) after bare-metal scaffold implantation, which recovers in about 2 weeks. Figure 8 As shown, this suggests local inflammatory infiltration caused by acute surgery. Two weeks later, the tracheal mucosa in the BNC-covered area still showed minimal CD45 infiltration, indicating that BNC-induced immune cell infiltration was minimal and did not trigger a sustained inflammatory effect.

[0106] (2) In vivo effects of PTX-BNC stent

[0107] In the PTX-BNC membrane-covered area, no obvious β-tubulin staining was observed, while CD45 staining was concentrated, suggesting local mucosal necrosis and inflammatory cell infiltration. Similar results were observed in the perimembrane area. Figure 7 The C-cell contrast study showed differences in two immunostaining assays among the three groups. This suggests that PTX is effectively released in vivo to exert its cytotoxic effect.

[0108] III. Conclusion

[0109] This invention utilizes the unique ability of *Acetobacter xylinum* to produce biocellulose (BNC) under aerobic conditions. By releasing oxygen through a metal scaffold loaded with CaO2 particles, the bacteria colonize the scaffold, gradually generating biocellulose. This ultimately achieves 360-degree encapsulation of the metal network by the biocellulose, constructing an integrated coated scaffold that enables drug loading and sustained release. This integrated coated scaffold exhibits no significant change in mechanical properties compared to the bare scaffold after 100 cycles of compression; and even at 90% compression, the stress is approximately 3.5N, maintaining high integration. Further in vivo animal experiments showed that two weeks after implantation into the trachea of ​​New Zealand white rabbits, tracheal patency was 100%, tracheal epithelial cells in the coated area survived, and no significant inflammatory cell infiltration or goblet cell metaplasia was observed. Finally, based on clinical data and 3D printing modeling, a customized coating scheme was designed on the metal scaffold. Porcine tracheal simulation experiments showed that the customized coated scaffold loaded with FITC-PTX can achieve precise drug delivery. In summary, this integrated covered scaffold constructed from microbial in-situ synthesized nanocellulose possesses high integration, large-capacity drug loading, good biocompatibility, and in vivo safety, making it a highly promising covered scaffold platform applicable to various intracavitary interventional therapies.

[0110] Comparative Example 1

[0111] Referring to the method in Example 1, an oxygen-free method was examined, in which a bare metal scaffold was directly placed in a mold for fermentation to prepare a BNC coated scaffold. After fermentation, the coated scaffold was removed. The results showed that this method easily caused the membrane to separate from the scaffold, making it difficult to achieve integrated preparation.

Claims

1. A method for preparing an integrated microbial in-situ synthetic membrane scaffold, characterized in that, Includes the following steps: (1) CaO2 nanoparticles were mixed with GelMA hydrogel solution, and then the metal scaffold was immersed in the hydrogel solution; after being taken out, it was cured under ultraviolet light to obtain a metal scaffold loaded with CaO2 nanoparticles. (2) Inoculate *Acetobacter xylosinophila* into the fermentation medium, shake and culture to obtain the activated bacterial culture solution; (3) A reactor is prepared by fixing a metal scaffold loaded with CaO2 nanoparticles between two silicone tubes with different inner and outer diameters. The fermentation broth filled with activated bacteria obtained in step (2) is inoculated into the sterilized reactor and placed at 30°C for static culture for 14 days to obtain a nanocellulose-coated scaffold. After removing the reactor, the nanocellulose-coated scaffold is purified to obtain the integrated coated scaffold.

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

3. The method according to claim 1, characterized in that, The metal support mentioned in step (1) includes any one of nickel-titanium alloy support, cobalt-chromium alloy support, 316L stainless steel support, nickel support, tantalum support, magnesium support, and zinc support.

4. The method according to claim 1, characterized in that, The curing process described in step (1) involves irradiating the product with 365 nm ultraviolet light for 5 minutes.

5. The method according to claim 1, characterized in that, The fermentation medium in step (2) is formulated as follows: 100 g / L D-fructose, 5 g / L peptone and 3 g / L yeast extract, with a pH of 5.

0.

6. The method according to claim 1, characterized in that, The sources of *Acetobacter xylinum* mentioned in step (2) include strains preserved on slant agar, strains preserved in cryopreservation solution, and bacterial cellulose membranes containing the strains. The inoculum size for strains preserved on slant agar is 1-2 loops, the inoculum size for strains preserved in cryopreservation solution is 1-5% (v / v), and the inoculum size for bacterial cellulose membranes containing the strains is 1-5 mm. 3 1-5 pieces of bacterial cellulose membrane.

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

8. The method according to claim 1, characterized in that, The inner and outer diameters of the two silicone tubes mentioned in step (3) are adjustable. The inner and outer diameters of the inner and outer silicone tubes range from 1.5-30 mm to 2-35 mm, and the difference between the inner diameters of the outer and inner silicone tubes is 0.5-10 mm.

9. The integrated covered scaffold prepared by the method according to any one of claims 1-8.

10. The application of the integrated covered scaffold of claim 9 in the preparation of tissue engineering scaffold materials.

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