Absorbable multifunctional Janus structure urethrostenosis repair material and preparation method thereof

Abstract

The invention provides an absorbable multifunctional Janus structure urethrostenosis repair material and a preparation method thereof, and belongs to the technical field of medical materials, the absorbable multifunctional Janus structure urethrostenosis repair material comprises a hydrophilic layer and a hydrophobic layer which are tightly connected; the hydrophilic layer is prepared from polyvinylpyrrolidone through electrostatic spinning; the hydrophobic layer is prepared from polycaprolactone through electrostatic spinning; the hydrophilic layer and the hydrophobic layer are tightly connected through a transition structure formed by electrostatic spinning at the same time; each layer is loaded with drugs, the hydrophilic layer is in contact with in-vivo tissues and can release anti-inflammatory drugs and growth factors and promote urethral tissue repair, and the hydrophobic layer is used for isolating urine, so that the urine is discharged smoothly and wound infection is prevented. The urethrostenosis repair material provided by the invention can improve the problem of urethrostenosis recurrence caused by urethral epithelium squamous metaplasia, fibrosis, inflammatory hyperplasia and the like easily caused by the existing urethrostenosis treatment means, and reduces the recurrence rate of urethrostenosis.

Description

Technical Field

[0001] The invention belongs to the technical field of medical materials, and particularly relates to an absorbable multifunctional Janus structure urethral stricture repair material and a preparation method thereof. Background Art

[0002] Recurrent urethral stricture has long been a challenge for urology both domestically and internationally, with no definitive and effective treatment options to date. Studies have shown that metal stents have achieved some success in treating urethral strictures, but long-term stent placement can irritate the urethra, leading to squamous metaplasia, fibrosis, and inflammatory hyperplasia of the urethral epithelium, which can cause recurrence of urethral strictures. Furthermore, proliferating granulation tissue caused by stent stimulation can protrude through the stent mesh into the urethra, causing re-obstruction. Therefore, inhibiting the proliferation of scar granulation tissue is key to treating restenosis after long-term stent placement.

[0003] Currently, metal stents have achieved some success in treating urethral strictures. However, their long-term placement can irritate the urethra, leading to squamous metaplasia, fibrosis, and inflammatory hyperplasia of the urethral epithelium, which can cause recurrence of urethral strictures. Furthermore, the proliferation of granulation tissue caused by stent irritation can penetrate the stent mesh and enter the urethra, causing re-obstruction. Therefore, inhibiting the proliferation of scar granulation tissue is key to treating restenosis after long-term stent placement.

[0004] Studies have investigated the use of drug-eluting stents to treat urethral strictures. Commonly used anti-restenotic drugs include cell cycle inhibitors, such as paclitaxel. These drugs effectively inhibit restenosis by disrupting the terminal pathways of intimal hyperplasia. Urethral reconstruction is often necessary for long-segment urethral strictures, but the use of alternative materials remains a challenge. Currently, urethral reconstruction primarily utilizes autologous tissue materials, such as foreskin flaps and tongue mucosa. However, the harvesting of these autologous tissues can be associated with donor-site complications and prolonged surgical and recovery times. Tissue engineering techniques offer a potential solution to these problems. Acellular collagen matrices (such as bladder acellular matrices and small intestinal acellular matrices) and polymeric materials synthesized from lactic acid-glycolic acid copolymers can serve as scaffolds for urethral reconstruction. However, the choice of these materials remains controversial. Acellular collagen matrices are derived from xenobiotics and may contain xenobiotics, leading to inflammatory reactions and the risk of infectious pathogens. Most synthetic polymeric materials produce acidic degradation products, which are detrimental to the growth of surrounding cells and can trigger a strong inflammatory response.

[0005] Polymers with excellent biocompatibility have been widely used in the reconstruction and repair of various organs and tissues, including bone, skin, and blood vessels. Domestic studies have used pure silk fibroin to repair 1.5 cm canine urethral defects, achieving relatively satisfactory results. However, when repairing urethral defects up to 3.0 cm, experimental animals commonly experienced complications such as urinary fistulas or urethral strictures. Furthermore, studies have successfully constructed tissue-engineered oral mucosa using BAM composite oral epithelial cells and effectively repaired 2 cm rabbit urethral defects. Other researchers have used the synthetic polymer PLGA composite urinary tract transitional epithelial cells to repair long-segment urethral strictures in beagle dogs. Electrospun silk fibroin, after stretching, exhibits good tissue compatibility with urinary tract transitional epithelial cells. Therefore, absorbable core-shell nanofibers are considered a potential material for urethral tissue engineering repair. Summary of the Invention

[0006] The purpose of the present invention is to provide an absorbable multifunctional Janus structure urethral stricture repair material and its preparation method, aiming to improve the problem that existing urethral stricture treatment methods are prone to cause urethral epithelial squamous metaplasia, fibrosis and inflammatory hyperplasia, which may lead to recurrence of urethral stricture, and reduce the recurrence rate of urethral stricture.

[0007] The present invention provides an absorbable multifunctional Janus structure urethral stricture repair material, comprising a tightly connected hydrophilic layer and a hydrophobic layer; the hydrophilic layer is prepared from polyvinyl pyrrolidone by electrospinning; the hydrophobic layer is prepared from polycaprolactone by electrospinning; a transition structure is formed between the hydrophilic layer and the hydrophobic layer by simultaneous electrospinning so that they are tightly connected.

[0008] Preferably, both the hydrophilic layer and the hydrophobic layer are loaded with drugs, and the drugs are one or more of vascular endothelial growth factor (VEGF), recombinant human epidermal growth factor (hEGF), and aspirin (APC).

[0009] The preparation method of the absorbable multifunctional Janus structure urethral stricture repair material comprises the following specific steps: 1) Add polyvinylpyrrolidone to anhydrous ethanol and stir until dissolved. This is called Solution A. Add polycaprolactone to a mixed solvent of N,N-dimethylformamide and dichloromethane and stir until dissolved. This is called Solution B. The concentration of polyvinyl pyrrolidone in solution A is 10-14 wt %; the concentration of polycaprolactone in solution B is 8-12 wt %; and the volume ratio of N,N-dimethylformamide to dichloromethane in the mixed solvent is 1:4.

[0010] 2) Transfer equal volumes of solution A and solution B into syringes respectively; first, electrospin solution A to form a membrane. When the solution in the syringe is about to be exhausted, electrospin solution B on the opposite side simultaneously, so that the hydrophilic layer produced by solution A and the hydrophobic layer produced by solution B are intertwined. Electrospinning is completed after solution B is exhausted.

[0011] 3) vacuum drying the product obtained in step 2) at room temperature for 24 hours to obtain the absorbable multifunctional Janus structure urethral stricture repair material.

[0012] Preferably, the electrospinning conditions in step 2) are: voltage 15-20 kV, receiver speed 150 rpm.

[0013] Preferably, in step 2), aspirin is added to solution A at a concentration of 0.5 mg / mL and growth factor is added at a concentration of 2.5 μg / mL; aspirin is added to solution B at a concentration of 1.0 mg / mL.

[0014] Preferably, in step 2), the electrospinning time of solution A and solution B simultaneously is not less than 15 minutes; Beneficial effects of the present invention: The absorbable multifunctional Janus structure urethral stricture repair material obtained by the method of the present invention can inhibit the proliferation of scar granulation tissue, fight inflammation and promote wound healing. When used in the treatment of urethral stricture, it can effectively maintain urethral patency while providing the required environmental humidity for surgical wound healing. It can also relieve pain, prevent fibrotic healing and promote rapid wound healing. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a scanning electron microscope image of an absorbable multifunctional Janus structure urethral stricture repair material of the present invention.

[0016] Figure 2 This is a contact angle diagram of an absorbable multifunctional Janus structure urethral stricture repair material of the present invention.

[0017] Figure 3 This is a diagram showing the cell survival-promoting effect of an absorbable multifunctional Janus structure urethral stricture repair material of the present invention.

[0018] Figure 4 This is a diagram showing the use of an absorbable multifunctional Janus structure urethral stricture repair material of the present invention in promoting the healing of urethral mucosal burn wounds at the in vivo level. DETAILED DESCRIPTION

[0019] In order to make the purpose, technical solutions and advantages of the present invention more clear, the technical solutions of the present invention are described clearly and completely in the form of specific embodiments below. If no specific conditions are specified in the examples, the conventional conditions or the conditions recommended by the manufacturer shall be followed.

[0020] Example 1 This embodiment provides a method for preparing an absorbable multifunctional Janus structure urethral stricture repair material, comprising the following steps: Solution A: Mix 0.84 g of dry polyvinylpyrrolidone K90 with 5.16 g of anhydrous ethanol and stir overnight on an electric magnetic stirrer until the solution is clear and free of particles. Solution B: Mix 0.48 g polycaprolactone, 1.10 g N,N-dimethylformamide, and 4.42 g dichloromethane and stir on an electric magnetic stirrer overnight until the solution is clear and free of particles. Take 3 mg of aspirin, 10 μg of recombinant human epidermal growth factor, and 2.5 μg of vascular endothelial growth factor and add them to solution A and mix thoroughly; take 6 mg of aspirin and add it to solution B and mix thoroughly; Transfer 5 mL of solution A to a syringe dedicated to electrospinning, set the roller receiver speed to 150 rpm, and apply a high voltage of 15 kV to the metal needle. When about 0.5 mL of solution A remains in the syringe, transfer 5 mL of solution B to a new syringe dedicated to electrospinning, place it on the other side of the roller receiver, and apply a high voltage of 17 kV to the metal needle. Electrospin solutions A and B simultaneously to interweave the hydrophilic and hydrophobic layers. After solution A is exhausted, continue electrospinning with solution B until solution B is exhausted. The fiber membrane with Janus structure obtained by electrospinning is carefully removed, placed in a vacuum drying oven for 24 hours to remove the residual solvent in the fiber membrane; it is cut into a suitable size to obtain an absorbable multifunctional Janus structure urethral stricture repair material.

[0021] Example 2 This embodiment provides a method for preparing an absorbable multifunctional Janus structure urethral stricture repair material, comprising the following steps: Solution A: Mix 0.6 g of dry polyvinylpyrrolidone K90 with 5.4 g of anhydrous ethanol and stir overnight on an electric magnetic stirrer until the solution is clear and free of particles. Solution B: Mix 0.72 g polycaprolactone, 1.05 g N,N-dimethylformamide, and 4.23 g dichloromethane and stir on an electric magnetic stirrer overnight until the solution is clear and free of particles. Take 3 mg of aspirin, 8.5 μg of recombinant human epidermal growth factor, and 4 μg of vascular endothelial growth factor and add them to solution A and mix thoroughly; take 6 mg of aspirin and add it to solution B and mix thoroughly; Transfer 5 mL of solution A to a syringe dedicated to electrospinning, set the drum receiver speed to 150 rpm, and apply a high voltage of 15 kV to the metal needle. When about 0.5 mL of solution remains in the syringe, transfer 5 mL of solution B to a new syringe dedicated to electrospinning, place it on the other side of the drum receiver, and apply a high voltage of 17 kV to the metal needle. Electrospin solutions A and B simultaneously to interweave the hydrophilic and hydrophobic layers. After solution A is exhausted, continue electrospinning with solution B until solution B is exhausted. The fiber membrane with Janus structure obtained by electrospinning is carefully removed, placed in a vacuum drying oven for 24 hours to remove the residual solvent in the fiber membrane; it is cut into a suitable size to obtain an absorbable multifunctional Janus structure urethral stricture repair material.

[0022] Example 3 This embodiment provides a method for preparing an absorbable multifunctional Janus structure urethral stricture repair material, comprising the following steps: Solution A: Mix 0.72 g of dry polyvinylpyrrolidone K90 with 5.28 g of anhydrous ethanol and stir overnight on an electric magnetic stirrer until the solution is clear and free of particles. Solution B: Mix 0.60 g polycaprolactone, 1.08 g N,N-dimethylformamide, and 4.32 g dichloromethane and stir on an electric magnetic stirrer overnight until the solution is clear and free of particles. Take 3 mg of aspirin, 11 μg of recombinant human epidermal growth factor, and 1.5 μg of vascular endothelial growth factor and add them to solution A and mix thoroughly; take 6 mg of aspirin and add it to solution B and mix thoroughly; Transfer 5 mL of solution A to a syringe dedicated to electrospinning, set the drum receiver speed to 150 rpm, and apply a high voltage of 15 kV to the metal needle. When about 0.5 mL of solution remains in the syringe, transfer 5 mL of solution B to a new syringe dedicated to electrospinning, place it on the other side of the drum receiver, and apply a high voltage of 17 kV to the metal needle. Electrospin solutions A and B simultaneously to interweave the hydrophilic and hydrophobic layers. After solution A is exhausted, continue electrospinning with solution B until solution B is exhausted. The fiber membrane with Janus structure obtained by electrospinning is carefully removed, placed in a vacuum drying oven for 24 hours to remove the residual solvent in the fiber membrane; it is cut into a suitable size to obtain an absorbable multifunctional Janus structure urethral stricture repair material.

[0023] Effect verification: In this embodiment, a scanning electron microscope is used to collect microscopic morphological images of an absorbable multifunctional Janus structure urethral stricture repair material provided in the embodiment, such as Figure 1 shown.

[0024] Figure 1 ① is a scanning electron microscope image of Janus nanofibers of the repair material close to the urethral mucosal layer. It can be seen that the fibers formed by electrospinning are relatively uniform, smooth and straight, and the fibers have different random orientations.

[0025] Figure 1 ② is a scanning electron microscope image of the Janus nanofibers of the repair material away from the urethral mucosa. It can be seen that the fibers formed by electrospinning are more uniform, smooth and straight, and are thinner than the Janus nanofibers close to the urethral mucosa. The fibers have different random orientations.

[0026] The hydrophilicity and hydrophobicity of the absorbable multifunctional Janus structure urethral stricture repair material provided in the embodiment were measured using a contact angle analyzer. Figure 2 shown.

[0027] Specifically, a sample was cut into 1 mm×1 mm and placed on a test plate with a liquid volume of 2 μL, and the contact angle after dropping was measured.

[0028] Figure 2 ① is the contact angle test diagram of the Janus nanofibers when the repair material is close to the urethral mucosa. It can be seen that the repair material has hydrophilic properties when it is close to the urethral mucosa.

[0029] Figure 2 ② is the contact angle test diagram of the Janus nanofibers when the repair material is away from the urethral mucosa layer. It can be seen that the repair material has hydrophobic properties when it is away from the urethral mucosa layer.

[0030] Effect of an absorbable multifunctional Janus structure urethral stricture repair material on cell survival, such as Figure 3 shown.

[0031] Live and dead cell fluorescence staining experiments were performed using a Calcein-AM / PI double staining kit. The experimental groups were divided into a control group (simple HUC cell culture) and an experimental group (HUC cells + repair material), with three replicates per group. HUC cells in the logarithmic growth phase were cultured at 5×10 4Cells were seeded in 24-well plates at a density of 10 cells / well and cultured for 24 hours to allow attachment. The experimental group was then replaced with complete culture medium containing the repair material, while the control group was replaced with an equal volume of complete culture medium without the repair material and cultured for a further 48 hours. For staining, the culture medium was discarded, the cells were gently washed twice with PBS, and 500 μL of a staining solution containing 2 μM calcein-AM and 5 μM PI was added to each well. The cells were incubated in the dark for 30 minutes. Fluorescence microscopy was then used for observation.

[0032] Live cells produce green fluorescence because Calcein-AM enters the cells and is hydrolyzed by esterase, while dead cells show red fluorescence because PI is embedded in DNA due to damaged cell membranes.

[0033] The density of green fluorescent cells in the field of view of the experimental group was significantly higher than that of the control group, and the cell outlines were clear and evenly distributed; while the number of red fluorescent cells in the control group was larger, and cell fragments were visible in some areas.

[0034] An absorbable multifunctional Janus structure urethral stricture repair material has a significant effect in promoting the healing of urethral mucosal burn wounds at the in vivo level. Figure 4 shown.

[0035] A high-frequency electrosurgery was used to establish a urethral mucosal burn model in male New Zealand white rabbits. Three burns were performed along the axial direction of the urethra, with an interval of 0.5 cm between each burn. The injury length was approximately 0.5 cm, and the depth was based on visible exposure of the submucosal layer.

[0036] Male New Zealand white rabbits were divided into three groups. The first group served as the control group, establishing a urethral mucosal burn model in male New Zealand white rabbits without receiving any dressing treatment. The second group, establishing a urethral mucosal burn model in male New Zealand white rabbits, received a repair material without growth factors applied to the injured area. The third group, establishing a urethral mucosal burn model in male New Zealand white rabbits, received a repair material containing growth factors applied to the injured area.

[0037] On the 14th day after surgery, the animals were euthanized with an overdose of sodium pentobarbital. The injured urethral tissue was completely removed along the urethra and fixed with 4% paraformaldehyde for 24 h before paraffin embedding. 5 μm thick sections were prepared and stained with HE.

[0038] Compared with the model control group, the degree of neutrophil infiltration in the urethral tissue of the experimental group using the repair material was significantly reduced, and the structural integrity of the mucosal epithelium was significantly improved.

[0039] With the addition of growth factors, neutrophil infiltration showed a decreasing trend. At the same time, it was observed that the number of new epithelial cell layers increased, the submucosal collagen fibers were arranged more orderly, and the density of new blood vessels was significantly improved.

[0040] Repair materials can effectively reduce the inflammatory response after urethral mucosal injury and promote tissue repair; and the addition of growth factors can synergistically enhance the repair efficiency of the material and show concentration dependence.

[0041] Through the evaluation of multiple experimental results, a multifunctional absorbable Janus structure urethral stricture repair material was prepared, which has targeted therapeutic effects, significant healing effects, and no adverse reactions to experimental animals.

[0042] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention should be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

Claims

1. An absorbable multifunctional Janus structure urethral stricture repair material, characterized in that: The material comprises a hydrophilic layer and a hydrophobic layer; the hydrophilic layer is prepared from polyvinyl pyrrolidone by electrostatic spinning; the hydrophobic layer is prepared from polycaprolactone by electrostatic spinning; and a transition structure is formed between the hydrophilic layer and the hydrophobic layer by simultaneous electrostatic spinning so as to enable them to be tightly connected.

2. The absorbable multifunctional Janus structure urethral stricture repair material according to claim 1, characterized in that: The hydrophilic layer and the hydrophobic layer are both loaded with drugs, and the drugs are one or more of vascular endothelial growth factor, recombinant human epidermal growth factor, and aspirin.

3. The preparation method of the absorbable multifunctional Janus structure urethral stricture repair material according to claim 1, characterized in that: The specific steps of this method are as follows: 1) Add polyvinylpyrrolidone to anhydrous ethanol and stir until dissolved. This is called Solution A. Add polycaprolactone to a mixed solvent of N,N-dimethylformamide and dichloromethane and stir until dissolved. This is called Solution B. The concentration of polyvinyl pyrrolidone in solution A is 10-14 wt %; the concentration of polycaprolactone in solution B is 8-12 wt %; and the volume ratio of N,N-dimethylformamide to dichloromethane in the mixed solvent is 1:

4. 2) Equal volumes of solution A and solution B are transferred to syringes. Solution A is first electrospun to form a membrane. When the solution in the syringe is about to be exhausted, solution B is simultaneously electrospun on the opposite side, so that the hydrophilic layer produced by solution A and the hydrophobic layer produced by solution B are intertwined. Electrospinning is completed when solution B is exhausted. 3) vacuum drying the product obtained in step 2) at room temperature for 24 hours to obtain the absorbable multifunctional Janus structure urethral stricture repair material.

4. The preparation method of the absorbable multifunctional Janus structure urethral stricture repair material according to claim 3, characterized in that: The electrospinning conditions in step 2) are: voltage 15-20 kV, receiver speed 150 rpm.

5. The preparation method of the absorbable multifunctional Janus structure urethral stricture repair material according to claim 3, characterized in that: In step 2), aspirin was added to solution A at a concentration of 0.5 mg / mL and growth factor was added at a concentration of 2.5 μg / mL; aspirin was added to solution B at a concentration of 1.0 mg / mL.

6. The preparation method of the absorbable multifunctional Janus structure urethral stricture repair material according to claim 3, characterized in that: In step 2), the electrospinning of solution A and solution B is performed simultaneously for no less than 15 minutes.

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