Composite gel artificial auricle material based on fiber web substrate strengthening strategy and preparation method thereof

Abstract

The invention discloses a composite gel artificial auricle material based on a fiber web substrate strengthening strategy and a preparation method thereof. The preparation method comprises the following steps: (1) designing and preparing a transparent mold of the fiber net substrate and a normal auricle shape; (2) preparing a polylactic acid-glycolic acid copolymer (PLGA) fiber web through electrospinning, cutting the PLGA fiber web into a required shape, and fixing the PLGA fiber web in a mold after heat setting; (3) pouring a ColMA solution containing cartilage promoting factors and mesenchymal stem cells, and carrying out light curing to obtain a composite gel artificial auricle inner layer; and (4) carrying out chondrogenic differentiation culture on the composite material for 4-8 weeks, then fixing the composite material in the mold again, and carrying out photocuring on a ColMA gel protective layer on the surface layer of the composite material to obtain the composite gel artificial auricle material based on the fiber web substrate strengthening strategy. The gel artificial auricle material has a bionic internal structure and an attractive appearance, and a pre-model is provided for research, development and practical application of artificial auricle products.

Description

Technical Field

[0001] The invention relates to the field of tissue engineering and artificial auricle repair, and in particular to a composite gel artificial auricle material based on a fiber mesh substrate reinforcement strategy and a preparation method thereof. Background Art

[0002] The artificial auricle is a plastic and reconstructive medical device used to replace missing or damaged auricle structures. By using biocompatible materials, the artificial auricle can highly simulate the appearance of the real auricle, helping patients restore the image of their ears and improve their self-confidence. Despite significant results in aesthetic and functional restoration, related surgeries and implants still face some challenges, especially infection risks and postoperative complications. These problems not only impose a physical and psychological burden on patients, but also increase the workload of medical workers. At present, there is still a lack of efficient solutions to these problems.

[0003] Based on the above problems, with the development of tissue engineering and materials science, artificial auricles based on chondrocytes have gradually moved from the laboratory to clinical application. This type of artificial auricle uses tissue engineering technology to culture, amplify and shape the patient's own or donated chondrocytes, and finally makes a substitute with similar structure and function to the natural auricle. This technology can not only effectively restore the auricle in appearance, but also has excellent biocompatibility, significantly reducing the risk of rejection and postoperative infection. In addition, the artificial auricle based on chondrocytes combines biomaterials and 3D printing scaffold technology, and cooperates with a variety of cytokines to induce differentiation to form an auricle scaffold that is more suitable for the individual patient, providing an ideal environment for the attachment, growth and differentiation of chondrocytes. However, due to the limitations of material selection of artificial auricles, it is often difficult to balance the biocompatibility and strength of the material. Although too hard materials can maintain the mechanical morphology of the artificial auricle, they often cannot support the distribution of chondrocytes well; the material selection and structural design that can support chondrocyte differentiation will also have aesthetic problems caused by easy degradation and deformation; therefore, there is an urgent need for a material selection and design that can balance biocompatibility and mechanical structure to improve the production of artificial auricles.

[0004] Based on the above problems, the present invention combines a variety of advanced tissue engineering technologies, innovatively uses PLGA electrospinning as a cell-loaded scaffold, and combines the fiber mesh substrate that can be formed after the photocurable ColMA material loads cells. This type of structure retains strong tissue compatibility and good diffusivity while ensuring period strength, and can greatly improve the generation of necrotic cores in cartilage differentiation. In addition, the present invention uses a depth perception application in a mobile phone or tablet with a laser radar to perform 3D reconstruction of the auricle, and with a customizable fiber mesh structure, it can maximize the personalized customization of function and morphological beauty in the model design of residual ears and deformed ears, making it convenient for patients to communicate with doctors about their diagnosis and treatment needs. Summary of the invention

[0005] The purpose of the present invention is to provide a composite gel artificial auricle material based on a fiber mesh substrate reinforcement strategy and a preparation method thereof, in view of the fact that the prior art is limited by the material selection of artificial auricles, and it is often difficult to balance the biocompatibility and strength of the materials. Although the overly hard materials can maintain the mechanical morphology of the artificial auricles, they often cannot support the distribution of cartilage. The material selection and structural design that can support the differentiation of cartilages also have aesthetic problems caused by easy degradation and deformation. The present invention provides a composite gel artificial auricle material based on a fiber mesh substrate reinforcement strategy and a preparation method thereof. The gel artificial auricle pre-model has a bionic internal structure and an aesthetic appearance, and provides a pre-model for the research and development and practical application of artificial auricle products.

[0006] The present invention is implemented by the following technical solution, and the specific preparation steps are as follows:

[0007] A method for preparing a composite gel artificial auricle material based on a fiber mesh substrate reinforcement strategy comprises the following steps:

[0008] 1) Design and prepare a transparent mold of a fiber mesh substrate and a normal auricle morphology;

[0009] 2) stirring and dissolving the inner layer polylactic acid-glycolic acid copolymer (PLGA) to form an electrospinning solution, and combining the electrospinning process to obtain a PLGA fiber mesh; after the fiber mesh is dried and formed, it is cut according to the fiber mesh base structure and assembled according to the shape of a transparent mold, and then heat-set and fixed in the transparent mold to obtain a fiber mesh core layer;

[0010] 3) preparing a cell-loaded ColMA pre-solution; pouring the cell-loaded ColMA pre-solution into a mold, degassing it by low-power ultrasound after completion, and then shaping it by UV light curing to obtain an artificial auricle middle layer structure, which is then taken out of the mold and placed in a culture medium for directional differentiation into cartilage; thereby obtaining a fiber mesh base structure loaded with chondrocytes;

[0011] 4) preparing a ColMA pre-solution and cooling it, then placing the chondrocyte-loaded fiber mesh substrate structure into a mold, pouring the ColMA pre-solution, and using UV light curing to form a composite gel artificial auricle with a fiber mesh substrate reinforcement strategy.

[0012] Furthermore, in step 1), during the process of preparing the fiber mesh substrate and the transparent mold of the normal auricle morphology, a laser radar is used in combination with a depth perception application to reconstruct the auricle as needed (for example, to reconstruct a residual ear or a deformed ear), and a 3D model file is obtained. Based on the model file, a fiber mesh substrate structure and a transparent mold of the normal auricle morphology are designed in a modeling software; in the process of obtaining the 3D model file, the 3D modeling method includes one or more of a splash method, a Gaussian mixture model, and a surface mesh subdivision method;

[0013] The modeling software includes one or more of CAD, 3Dmax and blender.

[0014] Furthermore, in step 2), during the preparation of the electrospinning solution, the PLGA is one or more of PLGA 75:25 and PLGA 50:50, the solvent is one or more of chloroform, dichloromethane and N,N-dimethylformamide (DMF), and the PLGA concentration is 10% (w / v) to 20% (w / v);

[0015] During the electrospinning process, the voltage is 10-20 kV, the speed is 3-8 mL / h, the drum speed is 60-90 r / min, the receiving distance is 15-25 cm, and the spinning spacing is 100-500 μm;

[0016] In the drying and molding process, nitrogen drying or vacuum drying is performed;

[0017] During the heat setting process, the temperature is 50-60°C.

[0018] Furthermore, in step 3), during the preparation of the cell-loaded ColMA pre-solution, ColMA powder is dissolved in a PBS solution containing a photoinitiator under sterile and light-proof conditions and cooled to obtain a ColMA solution, and finally mesenchymal stem cells are dispersed in the ColMA solution to obtain a cell-loaded ColMA pre-solution;

[0019] The concentration of the ColMA solution is 5-7.5wt%, the dissolution temperature of the ColMA powder is 50°C-65°C, the added photoinitiator is one or more of LAP, Irgacure, and I2959, with a concentration of 0.1-0.25wt%, the cooling temperature is below 37°C, the generation number of mesenchymal stem cells is P2-P5, and the concentration is 5×10 6 Pieces / mL.

[0020] Furthermore, in step 3), the pouring process is: pouring and filling the mold along the wall of the mold liquid filling port;

[0021] The ultrasonic power of the low-power ultrasonic degassing is 80-100W;

[0022] During the UV curing process, the wavelength of the light source is 350-410nm and the power is 20-40mW / cm 2 , time is 20-30s;

[0023] The culture medium is one or more of low-glucose DMEM (Dulbecco's Modified Eagle Medium) or α-MEM (Minimum Essential Medium Alpha); the chondrogenic factors added to the culture medium are chondrogenic factors including: one or more of TGF-β3 (transforming growth factor β3), BMP-7 (bone morphogenetic protein 7), IGF-1 (insulin-like growth factor-1) and PDGF-AB (platelet-derived growth factor AB), with a concentration of 100-300 ng / ml;

[0024] The time for chondrogenic differentiation is 4-8 weeks.

[0025] Furthermore, in step 4), during the preparation of the ColMA pre-solution, the ColMA powder is dissolved in a PBS solution containing a photoinitiator under sterile and light-proof conditions, the solution concentration is 15-20wt%, the dissolution temperature is 50-65° C., and the photoinitiator is one or more of LAP, Irgacure, and I2959, and the concentration is 0.1-0.25wt%;

[0026] During the UV curing process, the wavelength of the light source is 350-410nm and the power is 20-40mW / cm 2 , the curing time is 20-30s.

[0027] A composite gel artificial auricle material based on a fiber mesh substrate reinforcement strategy is obtained by using any one of the preparation methods described above.

[0028] Compared with the prior art, the present invention has the following advantages:

[0029] 1) The present invention uses PLGA, ColMA and other bioactive materials as raw materials, loads mesenchymal stem cells, and combines electrospinning, heat setting, casting, step-by-step UV light curing and in vitro cartilage induced differentiation to prepare a composite gel artificial auricle material with a fiber mesh base reinforcement strategy. The raw material selection, process combination and system composition are innovative.

[0030] 2) The present invention uses laser radar combined with depth perception application to perform 3D modeling of the artificial auricle, replacing the perfusion molding and CT-based modeling methods in traditional solutions; compared with the perfusion molding modeling method, this type of 3D modeling method provides the possibility of customizing the artificial auricle morphology, and compared with the CT modeling method, this method greatly simplifies the modeling cost and difficulty, and the patient and the doctor can customize the aesthetics and function of the artificial auricle through direct communication. The above-mentioned modeling method for the artificial auricle is an original technical innovation of the present invention.

[0031] 3) The present invention innovatively designs a new type of artificial auricle material with a multi-layer structure, which constructs the PLGA fiber mesh core layer structure, the chondrogenic differentiation composite gel middle layer structure and the high-concentration ColMA gel protective outer layer structure from the inside to the outside. The PLGA fiber mesh core layer provides the main mechanical support for the artificial auricle; the chondrogenic differentiation composite gel middle layer structure gradually forms a cartilage tissue structure similar to the natural auricle through chondrogenic differentiation, and its loose structure and good biocompatibility provide guarantees for the long-term survival and differentiation of stem cells; the high-concentration ColMA gel protective outer layer structure further improves the mechanical support of the artificial auricle through high-concentration ColMA, and provides external protection, providing a complete auricle shape, and meeting the patient's demand for aesthetics. Such a three-layer artificial auricle structure forms a natural auricle-like material through mutual cooperation from the inside to the outside, which is the first of its kind in the present invention.

[0032] 4) The present invention uses PLGA material and combines it with an electrospinning process to form a fiber mesh structure. After cutting, it is used as the internal core of the entire artificial auricle reinforcement using a heat setting process. Then, a ColMA solution containing mesenchymal stem cells is poured into a designed mold, and electrospinning, heat setting, pouring and UV light curing processes are connected in series to achieve a new application of the old process to produce an artificial auricle. After light curing, this type of fiber mesh substrate has a higher strength, thereby supporting the chondrogenic differentiation of mesenchymal stem cells. This structure provides a new pre-model for chondrocyte-based artificial auricles.

[0033] 5) The invention innovatively uses a secondary molding process for artificial auricle formation. First, the fiber reinforcement strategy of PLGA material is combined with in vitro chondrogenic differentiation to achieve the formation of thick cartilage that plays a structural support role in the artificial auricle. Then, ColMA light curing technology is used to form a thinner ColMA gel to replace the thin cartilage and other tissues, and integrate the entire auricle structure. The artificial auricle structure formed by this technology has a more bionic mechanical structure and appearance. At the same time, the fiber mesh base structure in the thick cartilage can greatly enhance its permeability and improve its survival ability.

[0034] 6) In the present invention, artificial auricle morphological models of different sizes can be obtained by scanning and 3D reconstruction of the auricles of different patients. Therefore, the artificial auricle materials prepared can meet various physiological needs of different patients, and the technology has strong universality. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 A schematic diagram of the effect of using a laser radar of a mobile device combined with a depth perception application to model a human skull and auricle in the present invention;

[0036] Figure 2 The figure is a schematic diagram of the preparation process of the composite gel artificial auricle material based on the fiber mesh substrate reinforcement strategy of the present invention. DETAILED DESCRIPTION

[0037] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0038] Comparative Example 1:

[0039] 1) Artificial auricle shape and casting mold design: Figure 1 As shown, a device with a laser radar is used in combination with a depth perception application to perform 3D reconstruction of the patient's auricle and generate a 3D model file in OBJ format. The file is imported into the Blender software, and based on the model file and the natural auricle shape, a hydrogel substrate, i.e., a fiber mesh, is designed for the auricle cartilage part; in addition, a transparent polyethylene mold for casting the entire auricle structure is designed based on the outer dimensions of the auricle.

[0040] 2) Preparation of inner hydrogel substrate and chondrogenic differentiation: Under sterile, light-proof and 60°C heating conditions, ColMA powder was dissolved in PBS solution containing 0.5 wt% LAP to prepare a 5% ColMA solution. After cooling to 37°C, the cultured P2 mesenchymal stem cells were cultured at 5×10 6 The cells were dispersed in the ColMA solution at a concentration of 100 / mL to form a cell-carrying ColMA pre-solution. The pre-solution was degassed by low-power ultrasonication (ultrasonic power of 80W) and poured into a mass. The mass was then immediately washed with water at a wavelength of 410nm and a power of 20mW / cm 2 The hydrogel mass was then trimmed into the designed hydrogel base shape and cultured in low-glucose DMEM medium supplemented with 100 ng / ml TGF-β3, 50 ng / ml BMP-7 and 50 ng / ml IGF-1 for 8 weeks until the chondrocytes differentiated and matured, and the chondrocyte-loaded gel base structure was obtained.

[0041] 3) Molding of the outer layer of the composite gel artificial auricle: Under sterile, light-proof and 60°C heating conditions, ColMA powder was dissolved in a PBS solution containing 0.5wt% LAP to obtain a 15wt% ColMA pre-solution; after cooling to 37°C, the inner layer hydrogel that had completed cartilage differentiation was placed in a mold, the ColMA pre-solution was poured, and the wavelength was 410nm and the power was 20mW / cm 2 The composite gel artificial auricle was obtained by UV light curing for 20 seconds.

[0042] The composite gel artificial auricle was sliced ​​and the live-dead staining was used under a confocal microscope, and it was found that 89% of the cells were stained with green fluorescence and 11% were stained with red fluorescence. The glycosaminoglycans (GAGs) in the cartilage matrix were detected under an optical microscope after staining with Alcian blue, and it was found that the base part of the fiber network in the slice was stained with a uniform and dense blue, indicating that the cartilage was well differentiated. The bending strength test and tensile strength test of the artificial auricle showed that the composite gel artificial auricle was too soft and broke twice under the action of 5MPa force. Its strength is completely insufficient to meet the requirements for maintaining the appearance of the artificial auricle.

[0043] Embodiment 1:

[0044] 1) Design of fiber mesh base and artificial auricle mold: Use a device with laser radar and depth perception application to reconstruct the patient's auricle in 3D and generate a 3D model file in OBJ format. Import the file into Blender software, and based on the model file and the shape of the natural auricle, design the fiber mesh base structure in the auricle cartilage part and other parts that play a major supporting role, and design the size, cutting method and splicing method of the single-layer fiber mesh; in addition, design a transparent polyethylene mold for casting the entire auricle structure based on the outer size of the auricle, that is, a transparent mold of the normal auricle shape.

[0045] 2) Preparation of the inner polylactic acid-glycolic acid copolymer (PLGA) fiber mesh core layer: Dissolve PLGA50:50 powder at a concentration of 10% in sufficient chloroform at a temperature of 15°C, and dissolve at a stirring speed of 200r / min to form a uniform electrospinning solution. Place the prepared spinning solution in the electrospinning instrument, set the voltage to 10kV, the speed to 3mL / h, the drum speed to 60r / min, the receiving distance to 15cm, the spinning spacing to 100μm, and the spinning width to 1cm to produce an electrospun fabric. Dry the formed electrospun fabric in a vacuum, and repeat 3 times until the chloroform is completely evaporated; under sterile conditions, cut the electrospun fabric into a single-layer fiber mesh according to a preset shape, and then stack them together to form a pre-designed fiber mesh base shape. Place the entire fiber mesh base in an oven at 50°C for 20 minutes for heat setting. It is observed that the single-layer fiber mesh is tightly bonded, and the fiber mesh base structure is obtained;

[0046] 3) Preparation of the middle layer structure of composite gel artificial auricle and chondrogenic differentiation: Under sterile, light-proof and 60°C heating conditions, ColMA powder was dissolved in PBS solution containing 0.25wt% LAP to prepare a 5% ColMA solution. After cooling to 37°C, the cultured P2 mesenchymal stem cells were cultured at 5×10 6The concentration of cells / mL was dispersed in the ColMA solution to form a cell-carrying ColMA pre-solution; the pre-solution was poured into the mold along the mold liquid filling port, and after the solution completely immersed the mold, low-power ultrasonic degassing was performed with an ultrasonic power of 80W; and then immediately using a wavelength of 410nm and a power of 20mW / cm 2 The cell-carrying fiber mesh substrate was then peeled off from the mold, and after trimming the excess part, it was placed in a low-glucose DMEM medium supplemented with 100ng / ml TGF-β3, 50ng / ml BMP-7 and 50ng / ml IGF-1 and cultured for 8 weeks until the chondrocytes differentiated and matured, and the chondrocyte-carrying fiber mesh substrate structure was obtained.

[0047] 4) Forming of the outer structure of the composite gel artificial auricle with a fiber mesh substrate reinforcement strategy: Under sterile, light-proof and 60°C heating conditions, ColMA powder was dissolved in a PBS solution containing 0.25wt% LAP to prepare a 15% ColMA pre-solution; after cooling to 37°C, the fiber mesh substrate that had completed cartilage differentiation was placed in a mold, the ColMA pre-solution was poured, and the wavelength was 410nm and the power was 20mW / cm 2 The product was cured by UV light for 20 seconds to obtain a composite gel artificial auricle with a fiber mesh base reinforcement strategy.

[0048] Compared with Comparative Example 1, Example 1 uses a fiber mesh substrate made of PLGA electrospun fabric instead of a hydrogel substrate, which greatly improves the strength of the substrate structure. The composite gel artificial auricle was sliced, and the cells were found to be green fluorescent under a confocal microscope using live-dead staining, accounting for up to 92%, which indicates that the cells survive well in this type of substrate; using Alcian blue staining to detect glycosaminoglycans (GAGs) in the cartilage matrix under an optical microscope, it was found that the fiber mesh substrate part in the slice was stained with a uniform and dense blue, indicating that the cartilage was well differentiated. Using bending strength tests and tensile strength tests to test the strength of the artificial auricle, it was found that the composite gel artificial auricle with a fiber mesh substrate reinforcement strategy has a significant improvement in strength, and can withstand more than 10 bending times under a force of 5MPa, and its strength can meet the appearance maintenance of the artificial auricle.

[0049] Embodiment 2:

[0050] 1) Design of fiber mesh base and artificial auricle mold: Use a device with laser radar and depth perception application to reconstruct the patient's auricle in 3D and generate a 3D model file in OBJ format. Import the file into Blender software, and based on the model file and the shape of the natural auricle, design the fiber mesh base structure in the auricle cartilage part and other parts that play a major supporting role, and design the size, cutting method and splicing method of the single-layer fiber mesh; in addition, design a transparent polyethylene mold for casting the entire auricle structure based on the outer size of the auricle, that is, a transparent mold of the normal auricle shape.

[0051] 2) Preparation of the inner polylactic acid-glycolic acid copolymer (PLGA) fiber mesh core layer: Dissolve PLGA50:50 powder at a concentration of 10% in sufficient chloroform at a temperature of 15°C, and dissolve at a stirring speed of 200r / min to form a uniform electrospinning solution. The configured spinning solution is placed in the electrospinning instrument, and the voltage is set to 10kV, the speed is 3mL / h, the drum speed is 60r / min, the receiving distance is 15cm, the spinning spacing is 400μm, and the spinning width is 1cm to produce an electrospun fabric. The formed electrospun fabric is dried in a vacuum, and repeated 3 times until the chloroform is completely evaporated; under sterile conditions, the electrospun fabric is cut into a single-layer fiber mesh according to a preset shape, and then stacked together to form a pre-designed fiber mesh base shape. The entire fiber mesh base is placed in an oven at 50°C for 20 minutes for heat setting. It is observed that the single-layer fiber mesh is tightly bonded, and the fiber mesh base structure is obtained.

[0052] 3) Preparation of the middle layer structure of composite gel artificial auricle and chondrogenic differentiation: Under sterile, light-proof and 60°C heating conditions, ColMA powder was dissolved in PBS solution containing 0.25wt% LAP to prepare a 5% ColMA solution. After cooling to 37°C, the cultured P2 mesenchymal stem cells were cultured at 5×10 6 The concentration of cells / mL was dispersed in the ColMA solution to form a cell-carrying ColMA pre-solution; the pre-solution was poured into the mold along the mold liquid filling port and adhered to the wall. After the solution completely immersed the mold, low-power ultrasonic degassing was performed with an ultrasonic power of 80W, and then a wavelength of 410nm and a power of 20mW / cm 2 The cell-carrying fiber mesh substrate was then peeled off from the mold, and after trimming the excess part, it was placed in a low-glucose DMEM medium supplemented with 100ng / ml TGF-β3, 50ng / ml BMP-7 and 50ng / ml IGF-1 and cultured for 8 weeks until the chondrocytes differentiated and matured, and the chondrocyte-carrying fiber mesh substrate structure was obtained.

[0053] 4) Forming of the outer structure of the composite gel artificial auricle with a fiber mesh substrate reinforcement strategy: Under sterile, light-proof and 60°C heating conditions, ColMA powder was dissolved in a PBS solution containing 0.25wt% LAP to prepare a 15% ColMA pre-solution; after cooling to 37°C, the fiber mesh substrate that had completed cartilage differentiation was placed in a mold, the ColMA pre-solution was poured, and the wavelength was 410nm and the power was 20mW / cm 2 The product was cured by UV light for 20 seconds to obtain a composite gel artificial auricle with a fiber mesh base reinforcement strategy.

[0054] Compared with Example 1, Example 2 used a larger textile spacing of 400 μm when weaving the PLGA electrospun fabric. The composite gel artificial auricle was sliced ​​and found under a confocal microscope using live-dead staining. The cells were all green fluorescent, accounting for as high as 98%, which indicated that the cells survived well in this type of substrate; using Alcian blue staining to detect glycosaminoglycans (GAGs) in the cartilage matrix under an optical microscope, it was found that the fiber mesh base part in the slice was stained with a uniform and dense blue, indicating that the cartilage was well differentiated. Using bending strength tests and tensile strength tests to test the strength of the artificial auricle, it was found that compared with Example 1, the composite gel artificial auricle with a fiber mesh base reinforcement strategy with a larger textile spacing had a slightly lower strength. It could withstand about 6 bending times under a force of 5MPa before breaking, and its strength could basically meet the appearance maintenance of the artificial auricle.

[0055] Embodiment 3:

[0056] 1) Design of fiber mesh base and artificial auricle mold: Use a device with laser radar and depth perception application to reconstruct the patient's auricle in 3D and generate a 3D model file in OBJ format. Import the file into Blender software, and based on the model file and the shape of the natural auricle, design the fiber mesh base structure in the auricle cartilage part and other parts that play a major supporting role, and design the size, cutting method and splicing method of the single-layer fiber mesh; in addition, design a transparent polyethylene mold for casting the entire auricle structure based on the outer size of the auricle, that is, a transparent mold of the normal auricle shape.

[0057] 2) Preparation of the inner polylactic acid-glycolic acid copolymer (PLGA) fiber mesh core layer: Dissolve PLGA50:50 powder at a concentration of 10% in sufficient chloroform at a temperature of 15°C, and dissolve at a stirring speed of 200r / min to form a uniform electrospinning solution. The prepared spinning solution is placed in the electrospinning instrument, and the voltage is set to 10kV, the speed is 3mL / h, the drum speed is 60r / min, the receiving distance is 15cm, the spinning spacing is 100μm, and the spinning width is 1cm to produce an electrospun fabric. The formed electrospun fabric is dried in a vacuum, and repeated 3 times until the chloroform is completely evaporated; under sterile conditions, the electrospun fabric is cut into a single-layer fiber mesh according to a preset shape, and then stacked together to form a pre-designed fiber mesh base shape. The entire fiber mesh base is placed in an oven at 50°C for 20 minutes for heat setting. It is observed that the single-layer fiber mesh is tightly bonded, and the fiber mesh base structure is obtained.

[0058] 3) Preparation of the middle layer structure of composite gel artificial auricle and chondrogenic differentiation: Under sterile, light-proof and 60°C heating conditions, ColMA powder was dissolved in PBS solution containing 0.25wt% LAP to prepare a 5% ColMA solution. After cooling to 37°C, the cultured P2 mesenchymal stem cells were cultured at 5×10 6 The concentration of cells / mL was dispersed in the ColMA solution to form a cell-carrying ColMA pre-solution; the pre-solution was poured into the mold along the mold liquid filling port and adhered to the wall. After the solution completely immersed the mold, low-power ultrasonic degassing was performed with an ultrasonic power of 80W, and then a wavelength of 410nm and a power of 20mW / cm 2 The cell-carrying fiber mesh substrate was then peeled off from the mold, and after trimming the excess part, it was placed in a low-glucose DMEM medium supplemented with 100ng / ml TGF-β3, 50ng / ml BMP-7 and 50ng / ml IGF-1 and cultured for 2 weeks until the chondrocytes differentiated and matured, and the chondrocyte-carrying fiber mesh substrate structure was obtained.

[0059] 4) Forming of the outer structure of the composite gel artificial auricle with a fiber mesh substrate reinforcement strategy: Under sterile, light-proof and 60°C heating conditions, ColMA powder was dissolved in a PBS solution containing 0.25wt% LAP to prepare a 15% ColMA pre-solution; after cooling to 37°C, the fiber mesh substrate that had completed cartilage differentiation was placed in a mold, the ColMA pre-solution was poured, and the wavelength was 410nm and the power was 20mW / cm 2 The product was cured by UV light for 20 seconds to obtain a composite gel artificial auricle with a fiber mesh base reinforcement strategy.

[0060] Compared with Comparative Example 1, in Example 3, only mesenchymal stem cells were cultured for a short period of 2 weeks of chondrogenic differentiation. The composite gel artificial auricle was sliced, and the inner fiber mesh substrate was photographed using SEM. It was found that there was a loose square microporous structure of 100μm, and the network edge and central voids were evenly filled with cells; using live-dead staining under a confocal microscope, it was found that the cells were all green fluorescent, accounting for up to 92%, which indicated that the cells survived well in this type of substrate; using Alcian blue staining to detect glycosaminoglycans (GAGs) in the cartilage matrix under an optical microscope, it was found that only about 10% of the cells in the fiber mesh substrate in the slice were stained blue, indicating that the cartilage differentiation was poor. Using bending strength test and tensile strength test to test the strength of the artificial auricle, it was found that compared with Example 1, the number of bending times that can be sustained under a force of 5MPa exceeded 13 times, indicating that a shorter in vitro culture time results in a smaller degree of material degradation and better maintenance of mechanical properties.

Claims

1. A method for preparing a composite gel artificial auricle material based on a fiber mesh substrate reinforcement strategy, It is characterized in that The steps include: 1) Design and prepare a transparent mold of a fiber mesh substrate and a normal auricle morphology; 2) stirring and dissolving the inner layer polylactic acid-glycolic acid copolymer (PLGA) to form an electrospinning solution, and combining the electrospinning process to obtain a PLGA fiber mesh; after the fiber mesh is dried and formed, it is cut according to the designed fiber mesh base structure and assembled according to the shape of a transparent mold, and then heat-set and fixed in the transparent mold to obtain a PLGA fiber mesh core layer; 3) preparing a cell-loaded ColMA pre-solution; pouring the cell-loaded ColMA pre-solution into a mold, degassing it by low-power ultrasound after completion, and then curing it by UV light, then taking it out of the mold and placing it in a culture medium for directional cartilage differentiation; thereby obtaining a fiber mesh base structure loaded with chondrocytes, i.e., a middle layer structure of a chondrogenic differentiation composite gel; 4) preparing a ColMA pre-solution and cooling it, then placing the chondrocyte-loaded fiber mesh substrate structure into a mold, pouring the ColMA pre-solution, and using UV light curing to form a high-concentration ColMA gel to protect the outer layer structure, thereby obtaining a composite gel artificial auricle with a fiber mesh substrate reinforcement strategy.

2. A method for preparing a composite gel artificial auricle material based on a fiber mesh substrate reinforcement strategy according to claim 1, Features: In step 1), during the preparation of the fiber mesh substrate and the transparent mold of the normal auricle morphology, the auricle is reconstructed using a laser radar combined with a depth perception application to obtain a 3D model file, and the fiber mesh substrate structure and the transparent mold of the normal auricle morphology are designed in the modeling software based on the model file; in the process of obtaining the 3D model file, the 3D modeling method includes one or more of a splash method, a Gaussian mixture model and a surface mesh subdivision method, and the transparent mold is a polyethylene material; The modeling software includes one or more of CAD, 3Dmax and blender.

3. A method for preparing a composite gel artificial auricle material based on a fiber mesh substrate reinforcement strategy according to claim 1, Features: In step 2), during the preparation of the electrospinning solution, the PLGA is one or more of PLGA 75:25 and PLGA 50:50, the solvent is one or more of chloroform, dichloromethane and N,N-dimethylformamide (DMF), and the PLGA concentration is 10% (w / v) to 20% (w / v); During the electrospinning process, the voltage is 10-20 kV, the speed is 3-8 mL / h, the drum speed is 60-90 r / min, the receiving distance is 15-25 cm, and the spinning spacing is 100-500 μm; In the drying and molding process, nitrogen drying or vacuum drying is performed; During the heat setting process, the temperature is 50-60°C.

4. The method for preparing a composite gel artificial auricle material based on a fiber mesh substrate reinforcement strategy according to claim 1, Features: In step 3), during the preparation of the cell-loaded ColMA pre-solution, ColMA powder is dissolved in a PBS solution containing a photoinitiator under sterile and light-proof conditions and cooled to obtain a ColMA solution, and finally mesenchymal stem cells are dispersed in the ColMA solution to obtain a cell-loaded ColMA pre-solution; The concentration of the ColMA solution is 5-7.5wt%, the dissolution temperature of the ColMA powder is 50°C-65°C, the added photoinitiator is one or more of LAP, Irgacure, and I2959, with a concentration of 0.1-0.25wt%, the cooling temperature is below 37°C, the generation number of mesenchymal stem cells is P2-P5, and the concentration is 5×10 6 Pieces / mL.

5. The method for preparing a composite gel artificial auricle material based on a fiber mesh substrate reinforcement strategy according to claim 1, Features: In step 3), the pouring process is: pouring along the wall of the mold to fill the mold; The ultrasonic power of the low-power ultrasonic degassing is 80-100W; During the UV curing process, the wavelength of the light source is 350-410nm and the power is 20-40mW / cm 2 , time is 20-30s; The culture medium is one or more of low-glucose DMEM (Dulbecco's Modified Eagle Medium) or α-MEM (Minimum Essential Medium Alpha); the chondrogenic factors added to the culture medium are chondrogenic factors including: one or more of TGF-β3 (transforming growth factor β3), BMP-7 (bone morphogenetic protein 7), IGF-1 (insulin-like growth factor-1) and PDGF-AB (platelet-derived growth factor AB), with a concentration of 100-300 ng / ml; The time for chondrogenic differentiation is 4-8 weeks.

6. The method for preparing a composite gel artificial auricle material based on a fiber mesh substrate reinforcement strategy according to claim 1, Features: In step 4), during the preparation of the ColMA pre-solution, ColMA powder is dissolved in a PBS solution containing a photoinitiator under sterile and light-proof conditions, the solution concentration is 15-20wt%, the dissolution temperature is 50-65°C, and the photoinitiator is one or more of LAP, Irgacure, and I2959, with a concentration of 0.1-0.25wt%; During the UV curing process, the wavelength of the light source is 350-410nm and the power is 20-40mW / cm 2 , the curing time is 20-30s.

7. A composite gel artificial auricle material based on a fiber mesh substrate reinforcement strategy, It is characterized in that The method is adopted as claimed in any one of claims 1 to 6.

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