A hydrogel scaffold for urethral wound repair healing

Abstract

The application discloses a hydrogel support for urethral wound repair and healing, which comprises an antifouling surface layer, a ZEIN middle layer and a PNIPAM hydrogel bottom layer, wherein the antifouling surface layer is a cross-linked layer added with an antibacterial agent, and the PNIPAM hydrogel bottom layer is loaded with a repair drug. The application utilizes the characteristics that the temperature of urine is higher than that of the urethra, utilizes the temperature-sensitive characteristics of poly (N-isopropyl acrylamide), enables the drug loaded in the hydrogel to be released when the urine passes, is used for offsetting the negative effects caused by the tension change, and utilizes the synergistic effect of the three layers to accelerate the repair and healing of the urethral wound.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of hydrogel urethral stents, and particularly relates to a hydrogel stent for urethral wound repair and healing. BACKGROUND

[0002] Although biomimetic tissues and organs can be cultured in vitro, the biomimetic tissues and scaffolds manufactured using traditional methods such as electrospinning, rapid prototyping and freeze-drying cannot form a three-dimensional structure compared with natural tissues or organs. In addition, cells cannot be uniformly attached to the scaffold. Directly seeding cells on the scaffold can cause cell death, further causing necrosis of the tissue or organ. 3D printing is an emerging technology that has been widely used in the field of tissue engineering. Compared with traditional manufacturing techniques, it has made great progress, but there are still problems such as low cell seeding efficiency, uneven distribution and low spatial resolution.

[0003] The arginine-glycine-aspartic acid (RGD) sequence in the gelatin chain is beneficial to enhance cell attachment and promote cell growth. Sodium alginate (SA) and gelatin (Gel) as natural hydrogel materials have the advantages of biocompatibility, low cost, and printability, and have been widely and successfully applied in tissue engineering, especially in bone and skin tissue engineering. However, as a product of collagen hydrolysis, although the gelatin has cell adhesion sites on the molecular chain, the cross-linking of the gelatin hydrogel is formed by intermolecular hydrogen bonds. At physiological temperature (37℃), this hydrogen bond cross-linking is unstable and can be easily destroyed, resulting in a too fast degradation rate that cannot match the growth rate of the tissue. In order to improve the mechanical strength of the hydrogel, a large amount of work and research has been carried out, and some effective methods have been developed, such as chemical modification, composite of multiple materials, etc.

[0004] It has been found that graphene-based materials are a very promising reinforcing nanomaterial. Because they have antibacterial properties, angiogenic potential, high mechanical strength and low cytotoxicity. Studies have shown that adjusting the oxygen content on the surface of rGO can enhance cell adhesion and proliferation. The incorporation of reduced graphene oxide (rGO) increases the concentration of reactive oxygen species (ROS), promoting cell proliferation and wound healing.

[0005] The prior art mainly uses 3D printing, loads cells or factors, and combines different material combinations for wound repair. The closest prior art uses a polyacrylamide hydrogel cross-linked by ultraviolet light to repair the skin. SUMMARY

[0006] The present application aims to solve the above technical problems and improve the use effect.

[0007] The application designs a hydrogel support for urethral wound repair and healing, which comprises an anti-fouling surface layer, a ZEIN middle layer and a PNIPAM hydrogel bottom layer.

[0008] By adopting the above technical scheme, the poly(N-isopropyl acrylamide) has excellent expansion performance and can bring certain tension to stimulate the early repair of the urethra. In the long-term damage repair process, the stimulation of the tension change caused by the urethral expansion will affect the repair effect when the urine passes. By utilizing the feature that the temperature of the urine is higher than that of the urethra and the temperature-sensitive property of the poly(N-isopropyl acrylamide), the drug loaded in the hydrogel can be released when the urine passes, so as to offset the negative effect caused by the tension change.

[0009] Among them, the photo-crosslinked hydrogel with the strongest anti-fouling performance of the surface layer of the hydrogel can avoid bacterial and protein adhesion to pollute the wound; the middle layer of the hydrogel has good mechanical strength, and the natural protein with high strength is used for force transmission; the temperature-sensitive hydrogel of the bottom layer has high expansion capacity, can support the bottom to promote skin growth, and can be added with cytokines for regulation.

[0010] Preferably, the preparation steps of the PNIPAM hydrogel bottom layer are as follows: weighing and dissolving N-isopropyl acrylamide monomer, ammonium sulfate and N,N'-methylene bisacrylamide in water according to the proportion, adding urethral wound repair drugs according to the concentration ratio as needed, and finally adding a certain proportion of the accelerator tetramethyl ethylenediamine to form the PNIPAM hydrogel bottom layer.

[0011] Preferably, the preparation steps of the anti-fouling surface layer are as follows: weighing N,N-diethyl-2-acrylamide, N-hydroxyethyl acrylamide, N,N'-methylene bisacrylamide and lithium phenyl-2,4,6-trimethyl benzoyl phosphinate, and crosslinking under ultraviolet irradiation to form the anti-fouling surface layer.

[0012] Preferably, the preparation steps of the ZEIN middle layer are as follows: weighing and dissolving corn protein in an ethanol solution, plasticizing in the aqueous phase, drying in an oven, and forming the ZEIN middle layer.

[0013] Preferably, the components of the anti-fouling surface layer include N,N-diethyl-2-acrylamide, N-hydroxyethyl acrylamide, N,N'-methylene bisacrylamide and lithium phenyl-2,4,6-trimethyl benzoyl phosphinate.

[0014] Preferably, the components of the PNIPAM hydrogel bottom layer include poly(N-isopropyl acrylamide), ammonium sulfate and N,N'-methylene bisacrylamide.

[0015] Preferably, the PNIPAM hydrogel bottom layer component includes a promoter tetramethyl ethylenediamine.

[0016] Preferably, the ZEIN middle layer main component is zein.

[0017] The hydrogel scaffold designed for urethral wound repair and healing has the following beneficial effects:

[0018] 1. The antifouling surface layer, ZEIN middle layer and PNIPAM hydrogel bottom layer used as a three-layer hydrogel scaffold for urethral wound repair and healing, wherein the PNIPAM hydrogel bottom layer has excellent swelling performance, can bring a certain tension to stimulate the early repair of the urethra, and simultaneously utilizes the feature that the temperature of urine is higher than that of the urethra, according to the temperature-sensitive properties of the PNIPAM hydrogel bottom layer, the drug loaded in the hydrogel can be released when the urine passes, for offsetting the negative effects caused by the change in tension.

[0019] 2. The hydrogel surface layer in the present application is a photocrosslinked hydrogel with the strongest antifouling performance, which can avoid bacterial and protein adhesion and contamination of the wound, thereby reducing the infection of viruses or bacteria during the urethral wound repair and healing process.

[0020] 3. The hydrogel middle layer in the present application is zein, which has good mechanical strength, and the use of natural protein with high strength for mechanical transmission can reduce secondary injury of the wound during the urethral wound repair and healing process. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 is the infrared spectrum of the three-layer material in the hydrogel scaffold;

[0022] Figure 2 is the scanning electron microscope of the three-layer material in the hydrogel scaffold;

[0023] Figure 3 is a schematic diagram of the hydrogel scaffold in the biological urethra for repair;

[0024] Figure 4 is a histological comparison experiment diagram of the hydrogel scaffold in Example 1, Comparative Example 1 and Comparative Examples 2 and 3;

[0025] Figure 5 is an effect diagram of the hydrogel scaffold in Example 1, Comparative Example 1 and Comparative Example 3 on urethral repair;

[0026] Figure 6 is a biocompatibility experiment diagram of the hydrogel scaffold in Example 1, Comparative Example 1 and Comparative Example 3;

[0027] Figure 7is the anti-fouling effect comparison chart of the anti-fouling surface layer in the hydrogel support;

[0028] Figure 8 is the nanoindentation chart of the ZEIN middle layer in the hydrogel support;

[0029] Figure 9 is the volume expansion chart of the PNIPAM hydrogel bottom layer in the hydrogel support;

[0030] Figure 10 is the DSC curve (differential scanning calorimetry) chart of the PNIPAM hydrogel bottom layer in the hydrogel support. DETAILED DESCRIPTION

[0031] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art belong to the scope of protection of the present application.

[0032] Raw materials:

[0033] N,N-diethyl-2-propenamide: CAS number 2675-94-7, molecular weight: 127.18, purity > 98.0%

[0034] N-hydroxyethyl acrylamide: CAS number 7646-67-5, molecular weight: 115.13, purity > 98.0%

[0035] N,N'-methylenebisacrylamide: CAS number 110-26-9, molecular weight: 154.17, purity > 98.0%

[0036] Lithium phenyl-2,4,6-trimethylbenzoylphosphinate: CAS number 85073-19-4, molecular weight: 296.23, purity > 97.0% Zein: CAS number 9010-66-6, purity > 99.0%

[0037] Ethanol: CAS number 64-17-5, molecular weight: 46.07, purity > 95.0%

[0038] N-isopropyl acrylamide: CAS number 2210-25-5, molecular weight: 113.158, purity > 98.0%

[0039] Ammonium sulfate: CAS number 7783-20-2, molecular weight: 132.139, purity > 98.0%

[0040] Tetramethylethylenediamine: CAS number 110-18-9, molecular weight: 116.205, purity > 99.0%.

[0041] Unless otherwise specified, all of the above materials are commercially available raw materials.

[0042] Example

[0043] Example 1:

[0044] Preparation of the surface layer of S1 hydrogel: Weigh 10%wt of N,N-diethyl-2-acrylamide, 10%wt of N-hydroxyethylacrylamide, 1%wt of N,N′-methylenebisacrylamide and 1%wt of phenyl-2,4,6-trimethylbenzoyl lithium phosphinate, and crosslink them by irradiation with a 365nm wavelength, 10W ultraviolet lamp for 10s at room temperature to form the surface layer of hydrogel;

[0045] Preparation of the ZEIN middle layer of S2 hydrogel: 10% wt zein was dissolved in ethanol solution, transferred to the aqueous phase and plasticized, and dried in an oven at 70℃ for 24 hours to form the middle layer of the hydrogel.

[0046] Preparation of PNIPAM substrate for S3 hydrogel: Weigh 1.5g of N-isopropylacrylamide monomer, 0.05g of ammonium sulfate and 0.05g of N,N′-methylenebisacrylamide and dissolve them in 40ml of water according to the ratio. Add 200μl of 1mg / ml verteporfen (YAP inhibitor) according to the ratio. Finally, add 1%wt of accelerator tetramethylethylenediamine to form the PNIPAM substrate.

[0047] Synthesis of S4 hydrogel scaffold: The surface layer, middle layer, and bottom layer of hydrogel are stacked sequentially.

[0048] like Figure 1 The image shows the infrared characterization of each layer of the hydrogel scaffold material, as well as... Figure 2 The images show scanning electron microscope (SEM) images of each layer of the material. In this embodiment, the hydrogel scaffold employs a three-layer structure, and the beneficial effects are achieved through the synergistic effect of the three layers. The top layer is antifouling to prevent bacterial adhesion and infection; the middle layer provides mechanical support; and the bottom layer has thermosensitive properties and releases medication during urination to counteract the negative effects of tension changes. Combined with... Figure 3 The embodiments of this application adopt Figure 3 The procedures shown were used in animal experiments to achieve the effect of urethral wound repair and healing.

[0049] Comparative Example

[0050] Comparative Example 1

[0051] The difference between this comparative example and Example 1 is that hydrogel is used as a bio-ink material for the scaffold according to the prior art, and the hydrogel scaffold is constructed by 3D printing technology.

[0052] Comparative Example 2

[0053] The present comparative example differs from Example 1 in that a double-layered hydrogel scaffold is formed using a stain-resistant surface layer and a ZEIN middle layer.

[0054] Comparative Example 3

[0055] The present comparative example differs from Example 2 in that a three-layered hydrogel scaffold is formed using a stain-resistant surface layer, a ZEIN middle layer, and a PNIPAM hydrogel bottom layer, and the PNIPAM hydrogel bottom layer is not loaded with a drug.

[0056] Result analysis:

[0057] A comparative experiment was performed to obtain Comparative Example 1 according to the prior art, in which a hydrogel was used as a scaffold for a bio-ink material and was constructed by 3D printing technology. In addition, Comparative Example 2 and Comparative Example 3 were obtained according to variations of Example 1, which were comparative experiments of Example 1. The experimental animals were rabbits specially used for experiments.

[0058] Reference Figure 4 The figure shows a histological comparison experiment of a hydrogel scaffold, in which control is Comparative Example 1, which is directly closed without repair after injury modeling; 2L is Comparative Example 2, in which Comparative Example 2 is a double-layered hydrogel scaffold formed using a stain-resistant surface layer and a ZEIN middle layer; 3L is Comparative Example 3, in which Comparative Example 3 is a three-layered hydrogel scaffold formed using a stain-resistant surface layer, a ZEIN middle layer, and a PNIPAM hydrogel bottom layer; and 3L-VP is Example 1, in which Example 1 is a three-layered hydrogel scaffold formed using a stain-resistant surface layer, a ZEIN middle layer, and a PNIPAM hydrogel bottom layer, in which the PNIPAM hydrogel bottom layer is loaded with a drug. As can be seen from the figure, the 3L-VP wound healing has less scar formation, normal epithelial thickness, and good healing.

[0059] In Figure 5 The first row of experiments shows Comparative Example 1, the second row of experiments shows Comparative Example 3, and the third row of experiments shows Example 1.

[0060] By comparing Comparative Example 1 and Comparative Example 2, when a three-layer structure is used in the hydrogel scaffold and simultaneously has a stain-resistant surface layer, a ZEIN middle layer, and a PNIPAM hydrogel bottom layer, the effect is stronger than the effect of the current bio-ink material constructed by 3D printing technology to form a hydrogel scaffold.

[0061] By comparing Comparative Example 2 and Example 1, when the PNIPAM hydrogel bottom layer is loaded with a drug, it has a better effect on urethral wound repair and healing. The temperature-sensitive property of poly(N-isopropylacrylamide) is used to release the drug loaded in the hydrogel when urine passes through, which is used to offset the negative effects of tension changes.

[0062] And the X-ray imaging technology to get the experimental rabbits X-ray film clearly see, using the three-layer hydrogel scaffold in example 1 obtained the best urethral repair effect.

[0063] Referring to Figure 6 , using the comparative example 1 and comparative example 3 as comparative experiment of example 1, get the biocompatibility experiment diagram of hydrogel scaffold material. Among them, the green in the figure is the living cells, and the red is the dead cells. From the figure, it can be seen that the three-layer hydrogel scaffold in example 1 has good biocompatibility, and will not cause damage to the cells due to its biological toxicity, and can play its biological performance, and then get the best urethral repair effect of example 1.

[0064] Referring to Figure 7 , the anti-fouling effect of the anti-fouling surface layer in the hydrogel scaffold, the anti-fouling surface layer effect of example 1 is better. And in the known literature has been disclosed, the application example 1 adopted is the optimal ratio to realize the anti-fouling function of the anti-fouling surface layer.

[0065] Referring to Figure 8 , the nano indentation diagram of ZEIN middle layer in example 1, it can be seen from the figure that the ZEIN middle layer has more excellent mechanical effect.

[0066] Referring to Figure 9 , the volume expansion diagram of PNIPAM hydrogel bottom layer in hydrogel scaffold in example 1, it can be seen from the figure that the PNIPAM bottom layer can be quickly expanded and shaped in 5 minutes, which is conducive to the release of drug in the PNIPAM bottom layer and makes the urethral wound can be quickly covered and expanded to improve the treatment effect.

[0067] Referring to Figure 10 , Figure 10 is the DSC curve diagram of PNIPAM hydrogel bottom layer in hydrogel scaffold, from the differential scanning calorimetry, it can be seen that the PNIPAM hydrogel bottom layer has temperature sensitive characteristics, which is conducive to the release of drug for self-control of urination to offset the adverse effects of urination, and the comprehensive analysis of the peak is shown in Figure 10 .

[0068] Application example

[0069] The application of the three-layer hydrogel support obtained in Example 1 in the urethra of an animal rabbit and good treatment effect in the urethra of the animal rabbit, through animal models for experimental physiology, experimental pathology and experimental treatment, play a role in the field of biological and medical research, and are expected to achieve better application in the human urethra and good development prospects in the future. And the drug-loaded support suitable for urethral injury repair is designed according to the structure and environment of the urethra, and the characteristics of the temperature-sensitive hydrogel are used to achieve the release of the drug through the automatic control of urination to offset the adverse effects brought by urination.

[0070] The present application is not limited to the above-mentioned best mode, and anyone can derive other various forms of products under the inspiration of the present application, but regardless of any change in shape or structure, any technical solution with the same or similar to the present application falls within the scope of the present application.

Claims

1. A hydrogel scaffold for urethral wound repair and healing, characterized in that, It consists of an antifouling surface layer, a ZEIN middle layer, and a PNIPAM hydrogel bottom layer. The antifouling surface layer is a cross-linked layer with added antibacterial agent, and the PNIPAM hydrogel bottom layer contains urethral wound repair medication. The PNIPAM hydrogel bottom layer is prepared according to the following steps: weigh out the specified weight parts of N-isopropylacrylamide monomer, ammonium sulfate and N,N′-methylenebisacrylamide and dissolve them in water in proportion, add urethral wound repair drugs as needed according to the concentration ratio, and finally add a certain proportion of the accelerator tetramethylethylenediamine to form the PNIPAM hydrogel bottom layer. The antifouling surface layer is prepared by the following steps: weighing a set number of N,N-diethyl-2-acrylamide, N-hydroxyethylacrylamide, N,N′-methylenebisacrylamide and phenyl-2,4,6-trimethylbenzoyl lithium phosphinate, and crosslinking them under ultraviolet light to form an antifouling surface layer. The middle layer of ZEIN is composed of zein.

Images