Artificial auricle material compounded with vascularization and cartilage double frameworks and preparation method of artificial auricle material

Abstract

The invention discloses an artificial auricle material compounded with a vascularization and cartilage double framework and a preparation method of the artificial auricle material. Comprising the following steps: 1) performing 3D modeling as required to obtain an artificial auricle macroporous stent model, and presetting an angiogenic differentiation framework on the artificial auricle macroporous stent model; 2) based on a multi-material multi-nozzle photocuring 3D printing technology, using a GelMA solution to print an artificial auricle macroporous stent, using a mesenchymal stem cell (MSCs)-loaded GelMA solution to print a vascularized framework in a framework, and after angiogenesis differentiation culture, obtaining an intermediate form artificial auricle compounded with the vascularized framework; and (3) introducing a ColMA solution loaded with MSCs into pores of the artificial auricle in the middle form by combining a multi-time dipping-photocuring method, and carrying out chondrogenic differentiation culture to obtain the artificial auricle material compounded with the vascularization and chondrogenesis dual frameworks at the same time. A vascularization framework and a cartilage framework are arranged in the stent at the same time, and a design normal form is provided for an artificial auricle with a complete physiological function.

Description

Technical Field

[0001] The present invention relates to the field of 3D photocuring printing and artificial auricle materials, and in particular to an artificial auricle material having a composite vascularized and cartilaginized dual frame and a preparation method thereof. Background Art

[0002] As a plastic surgery and repair medical device, artificial auricle is mainly used to replace lost or damaged auricle structure. Made of biocompatible materials, artificial auricle can achieve highly bionic visual effects, restore the appearance of the patient's ears, and enhance self-confidence. Although it has achieved certain results in terms of aesthetics and functional restoration, related surgeries and implants also face some problems, especially infection risks and postoperative complications, which will bring physical and psychological burdens to patients and increase the workload of doctors. 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 out of the laboratory and into the clinic. This type of artificial auricle uses tissue engineering technology to culture, amplify and shape the patient's own or donated chondrocytes, and finally makes an artificial auricle with a structure and function similar to the natural auricle. This technology can not only restore the auricle in appearance, but also has better biocompatibility, reducing the risk of rejection and postoperative infection. In addition, this type of artificial auricle is combined with biomaterials and 3D printed scaffolds, and a variety of cytokines are used to induce differentiation to form an auricle scaffold that is more suitable for individual patients, and provides excellent conditions for chondrocyte attachment, growth and differentiation. However, due to the thickness of the artificial auricle, the weak blood supply of the surrounding tissues cannot support the normal physiological functions of the intermediate cells, and hypoxic cell apoptosis occurs, forming a necrotic core. This phenomenon will accelerate the apoptosis and disintegration of this type of artificial auricle, thereby hindering its long-term survival in the body. Therefore, in order to balance the strength of the cartilage structure and its ability to survive in the body, adding a microvascular structure that is conducive to the exchange of substances between cells into the cartilage structure is a necessary condition for the long-term survival of cartilage implants such as artificial auricles in the body.

[0004] Based on the above problems, the present invention combines a variety of advanced tissue engineering technologies, and uses a preset structure for angiogenic differentiation based on the existing artificial auricle combined with 3D scaffolds and chondrocytes, and combines 3D light-stereolithography to print macroporous scaffolds for chondrogenic differentiation, thereby obtaining an artificial auricle with a dual framework of vascularization and chondrogenic differentiation, which completely improves the blood supply and material exchange of the cartilage in the scaffold, and provides a new paradigm for the design of artificial auricles that can be used in the body for a long time. In addition, the present invention can perform 3D reconstruction of the patient's auricle based on laser radar and depth perception applications, providing a more user-friendly artificial auricle modeling solution, which greatly improves the efficiency of doctor-patient communication and can meet the patient's aesthetic and functional needs for ear structure reconstruction based on artificial auricle to a great extent during the design stage. Summary of the invention

[0005] The purpose of the present invention is to provide an artificial auricle material with a vascularized and cartilaginized dual framework and a preparation method thereof in view of the deficiencies of the prior art and by combining the advantages of various materials and processes.

[0006] The present invention is implemented by the following technical solutions:

[0007] A method for preparing an artificial auricle material having a composite vascularized and cartilaginized dual framework comprises the following steps:

[0008] 1) Artificial auricle bracket design: 3D modeling can be performed as needed. For example, laser radar can be used in combination with depth sensing applications to perform 3D modeling, and the patient's auricle can be reconstructed to obtain an artificial auricle large hole bracket model, and a vascular differentiation framework can be preset in the model;

[0009] 2) Preparation of the intermediate form of the artificial auricle composited with a vascularized frame: Based on the multi-material multi-nozzle light-curing 3D printing technology, the artificial auricle macroporous scaffold is printed with GelMA solution, and the vascularized frame is printed inside the frame with GelMA solution containing mesenchymal stem cells (MSCs), and angiogenic differentiation culture (usually about 10 days) is performed to obtain the intermediate form of the artificial auricle composited with a vascularized frame;

[0010] 3) Preparation of artificial auricle based on double frame: Combined with multiple immersion-light curing method, ColMA solution containing MSCs is introduced into the pores of the intermediate-form artificial auricle frame, and chondrogenic differentiation culture is performed (usually for about 4-6 weeks) to obtain an artificial auricle material with a composite vascularized and chondrogenic double frame.

[0011] Further, in step 1), in the depth perception application used in the laser radar mobile device, the 3D modeling method includes one or more of the splash method, the Gaussian mixture model and the surface mesh subdivision method. The mesh support diameter of the meshed artificial auricle macroporous stent model is 300-500μm, and the channel structure diameter is 200-300μm; the preset angiogenic differentiation framework is a high-density and dispersed angiogenic stent in the macroporous stent, and the stent diameter is 500-700μm.

[0012] Furthermore, in step 2), the concentration of the GelMA solution for printing the artificial auricle scaffold is 15-20wt%, and the dissolution temperature is 60-80°C; the concentration of the GelMA solution carrying mesenchymal stem cells is 5-7.5wt%, and the dissolution temperature is 60-80°C, and after complete dissolution, it is cooled to 35-37°C; the photoinitiator added to the above two GelMA solutions is one or more of LAP, Irgacure, and I2959, with a concentration of 0.1-0.25wt%, and the dissolution is carried out under light-proof conditions. The MSCs are within the P2-P5 generations, with a concentration of 1×10 7 The concentration of 100 / mL was dispersed in the low concentration GelMA solution.

[0013] Furthermore, in step 2), the multi-material multi-nozzle light-curing 3D printer uses a dual-nozzle dual-tank structure, the light source is a 350-410nm UV light source, and the light source power is 20-40mW / cm 2 The nozzle diameter for printing the auricle pore scaffold is 300-500μm, the printing layer thickness is 300-500μm, and the layer interval is 200-300μm; the nozzle for printing the vascular differentiation framework is 500-700μm, and the printing layer thickness is 500-700μm; the printing temperature is 35℃-37℃, and the humidity is 60%-80%.

[0014] Furthermore, in step 2), the culture medium for angiogenic differentiation culture uses a basal culture medium of one or more of low-glucose DMEM or α-MEM, and the added angiogenic factors are one or more of VEGF, FGF-2, EGF and IGF-1, with a concentration range of 5-10 ng / ml.

[0015] Further, in step 3), the ColMA solution has a concentration of 5-7.5wt%, a dissolution temperature of 60-80°C, and is cooled to 35-37°C after complete dissolution; the photoinitiator is one or more of LAP, Irgacure, and I2959, with a concentration of 0.1-0.25wt%; the MSCs generation is P2-P5, and the concentration is 5×10 6 / ml; the basic culture medium for chondrogenic differentiation culture is one or more of low-glucose DMEM or α-MEM; the chondrogenic factors added in the ColMA solution and the culture medium mainly include the following: one or more of TGF-β, BMPs, IGF-1 and FGF-2, with concentrations of 10-20ng / ml and 5-10ng / ml respectively.

[0016] Furthermore, in step 3), the immersion time of the cyclic immersion-light curing method is 30-50 seconds, and the light source during light curing is a UV light source of 350-410 nm and the power is 20-40 mW / cm 2 , the irradiation time is 10-15s, and the number of cycles is 3-5 times.

[0017] Compared with the prior art, the present invention has the following advantages:

[0018] 1) The present invention uses bioactive materials such as ColMA and GelMA as raw materials, loads mesenchymal stem cells, and combines multi-nozzle 3D photocuring printing, cyclic immersion-photocuring method and in vitro cartilage induced differentiation and other combined processes to produce an artificial auricle structure with a dual frame of vascularization and cartilage. The raw material selection, process combination and system composition are innovative.

[0019] 2) The present invention uses laser radar combined with depth perception 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 shape of the artificial auricle, 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.

[0020] 3) The present invention is committed to retaining the mechanical structure of the chondrocyte artificial auricle while improving its long-term survival ability in the body. A triple gel structure is pre-designed during 3D modeling: a high-concentration GelMA macroporous framework provides a mesenchymal stem cell attachment base structure and a main mechanical support scaffold structure, and a low-concentration GelMA framework is designed in the above-mentioned high-concentration GelMA macroporous framework. Its mild mechanical environment loads mesenchymal stem cells, which can greatly promote vascular differentiation and greatly improve the nutrition supply and waste exchange of the artificial auricle. The triple ColMA-loaded mesenchymal stem cells are loosely distributed in the pores of the macroporous scaffold through immersion-photocuring, and can form an artificial auricle with good biological activity and rigid structure through chondrogenic differentiation. Through the synergistic effect of the above-mentioned structure, a natural auricle with vascular and cartilage structure can be highly simulated and reproduced, which has important clinical value and significance for promoting the development of artificial auricle.

[0021] 4) The artificial auricle material with a dual framework of vascularization and chondrogenesis in the present invention has the functions of angiogenesis and chondrogenesis, and can maintain the appearance structure of the auricle, providing aesthetic support. Through the innovative design of step-by-step differentiation, angiogenesis differentiation is completed within the framework, and then chondrogenesis differentiation is carried out. The mild mechanical environment formed by the low concentration of GelMA provides good conditions for the proliferation and differentiation of various cells, and the framework formed by the high concentration of GelMA not only provides good mechanical support, but its long degradation cycle also lays a good foundation for the long-term function of the artificial auricle in the body.

[0022] 5) The present invention integrates a preset vascular differentiation framework structure into a macroporous scaffold, and adopts a 3D photocuring printing process with dual nozzles and dual material troughs. Through this method, the vascular differentiation framework is formed and printed in the macroporous scaffold at one time. There are a large number of mesenchymal stem cells in the fibers formed by the vascular differentiation framework. During angiogenic differentiation, the mesenchymal stem cells will move to the surface of the fiber and form a tubular structure while differentiating on the surface. As the differentiation proceeds, the low concentration of GelMA in the scaffold will be degraded, and finally a loose vascular structure will be formed, which can provide a stronger blood supply formation ability when implanted in the body.

[0023] 6) The 3D modeling process and the artificial auricle structure with a composite vascularized and cartilaginized dual frame in the present invention are almost applicable to the technical requirements of various types of patients for artificial auricles and have extremely strong universality. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a schematic diagram of the preparation process of the artificial auricle material with a vascularized and cartilaginized dual framework in the present invention;

[0025] Figure 2 Schematic diagram of the artificial auricle scanning and reconstruction results. DETAILED DESCRIPTION

[0026] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. Figure 1 Shown is a schematic diagram of the process for preparing the artificial auricle material of the present invention.

[0027] Comparative Example 1:

[0028] 1) Design of artificial auricle support: Figure 2 As shown, the patient's auricle is 3D reconstructed using the depth perception function in the 3D reconstruction of the device with laser radar, and a 3D model file in OBJ format is generated. The file is imported into Blender software. Based on the model file, a gridded macroporous stent structure is designed, with a stent diameter of about 300 μm and a pore structure diameter of about 200 μm; the file is saved as an STL file for printing.

[0029] 2) Preparation of pre-printing solution: Under sterile, light-proof and 60°C heating conditions, the GelMA60 lyophilized powder was dissolved in a PBS solution containing 0.25 wt% LAP to prepare a 15 wt% high-concentration GelMA solution as a pre-printing solution for photocuring printing of the scaffold.

[0030] 3) Photocuring printing of artificial auricle frame: calibrate the 3D photocuring printing platform and load the STL model file. Load the pre-printing solution into the material tank and use high-concentration GelMA to print the artificial auricle frame; the nozzle diameter of the artificial auricle frame is 300μm, the layer spacing is 200μm, the light source is a 410nm UV light source, and the light source power is 20mW / cm 2 , printing temperature is 37℃ and humidity is 60%.

[0031] 4) Artificial auricle framework cartilage differentiation: Under sterile, light-proof and 60°C heating conditions, ColMA was dissolved in PBS containing 0.25 wt% LAP to prepare ColMA solution, and P3 mesenchymal stem cells were cultured at 5×10 6 The density of 1000 μg / mL was dispersed in it, and then 20 ng / ml TGF-β, BMPs, IGF-1 and FGF-2 were added to the ColMA solution. After the surface of the artificial auricle frame was dried, the solution was completely filled into the artificial auricle frame using the immersion-light curing method. After immersion for 30 seconds, a 410 nm UV light source with a light source power of 20 mW / cm 2 , irradiated for 10s, repeated three times. Then, the cells were cultured in low-glucose DMEM medium containing 10ng / ml TGF-β, BMPs, IGF-1 and FGF-2 for 6 weeks to obtain artificial auricle materials.

[0032] 5) The above artificial auricle was sliced, and live-dead staining was used to find a large number of clustered cells in the frame under a confocal microscope. Among the clustered cells, 41% had a large number of red fluorescence, and 59% had green fluorescence, indicating the presence of a large number of necrotic cores. The slices were stained with toluidine blue to mark chondrocytes. It was found under a confocal fiber microscope that no more than 15% of the cells were marked and were distributed in disorder in the slices. The above observation method suggests that this type of artificial auricle material has a low cell survival rate and low cartilage differentiation efficiency due to the low ability of internal mesenchymal stem cells to exchange nutrients and waste.

[0033] Embodiment 1:

[0034] 1) Artificial auricle bracket design: Use the depth perception function in the 3D reconstruction of the device with laser radar to perform 3D reconstruction of the patient's auricle and generate a 3D model file in OBJ format. Import the file into Blender software. Based on the model file, design a gridded macroporous bracket structure with a bracket diameter of about 300μm and a channel structure diameter of about 200μm; then design a high-density and dispersed vascular bracket inside the macroporous bracket with a bracket diameter of about 500μm; save the file as an STL file for printing.

[0035] 2) Preparation of printing pre-solution: Under sterile, light-proof and 60°C heating conditions, GelMA60 freeze-dried powder was dissolved in PBS solution containing 0.25wt% LAP, and 15wt% high-concentration and 5wt% low-concentration GelMA solutions were prepared respectively. P3 mesenchymal stem cells were cultured at a rate of 1×10 7 The concentration of cells / mL was dispersed in a low-concentration GelMA solution to obtain a low-concentration GelMA pre-solution containing cells and a high-concentration GelMA pre-solution without cells.

[0036] 3) Preparation of intermediate-form artificial auricle with vascularized framework: calibrate the 3D light-curing printing platform and load the STL model file. Load the low-concentration GelMA pre-solution containing cells and the high-concentration GelMA pre-solution without cells into the material tank, and use the double-nozzle double-tank structure installed in the light-curing 3D printer. The high-concentration GelMA solution prints the macroporous scaffold framework of the artificial auricle, and the low-concentration GelMA solution containing mesenchymal stem cells prints the preset vascularized scaffold (i.e., vascular framework); the nozzle diameter for printing the macroporous scaffold framework is 300μm, the layer spacing is 200μm, the nozzle diameter for printing the vascular framework is 500μm, the light source is a 410nm UV light source, and the light source power is 20mW / cm 2 , the printing temperature was 37°C and the humidity was 60%. After printing, the entire scaffold was cultured in a low-glucose DMEM medium containing 10ng / ml VEGF, FGF-2, EGF and IGF-1 for 10 days, and after the angiogenic differentiation was completed, an intermediate morphology artificial auricle with a vascularized framework was obtained;

[0037] 4) Preparation of artificial auricle based on double frame: ColMA solution was prepared by dissolving ColMA in PBS containing 0.25 wt% LAP under sterile, light-proof and 60°C heating conditions, and P3 mesenchymal stem cells were cultured at 5×10 6The density of 1000 μg / mL was dispersed in it, and then 20 ng / ml TGF-β, BMPs, IGF-1 and FGF-2 were added to the ColMA solution. After the surface of the intermediate-shaped artificial auricle was dried, the solution was completely filled into the entire auricle frame using the immersion-light curing method. After immersion for 30 seconds, a 410 nm UV light source with a light source power of 20 mW / cm 2 , irradiated for 10 seconds, repeated three times. Then, the cells were cultured in low-glucose DMEM medium containing 10 ng / ml TGF-β, BMPs, IGF-1 and FGF-2 for 6 weeks to obtain an artificial auricle material with a dual framework of vascularization and cartilage.

[0038] 5) Compared with Comparative Example 1, Example 1 presets a vascular framework in the artificial auricle large hole scaffold, and performs angiogenesis and chondrogenesis differentiation in sequence. The artificial auricle structure with a dual framework of vascularization and chondrogenesis was sliced, and live-dead staining was used to find that the cells were almost all green fluorescence under a confocal microscope, accounting for up to 89%, which suggests that the cells survive well in this type of substrate; the slices were stained with toluidine blue and von Willebrand factor (vWF) immunofluorescence staining to mark the chondrocytes and vascular endothelial cells of the artificial auricle with a dual framework of vascularization and chondrogenesis, respectively. It was observed under a confocal fiberscope that the number of chondrocytes uniformly labeled with blue dye accounted for up to 81%, while the vascular endothelial cells formed a uniform and continuous network structure, accounting for 8%. The above phenomenon suggests that the chondrocytes in the artificial auricle with a dual framework of vascularization and chondrogenesis are evenly and densely differentiated and present a good cartilage morphology; through the means of two-step differentiation, vascular endothelial cells also form a dense vascular network in the framework. Such a structure lays the most basic histological structural foundation for the long-term survival of this type of artificial auricle in the body.

[0039] Embodiment 2:

[0040] 1) Artificial auricle bracket design: Use the depth perception function in the 3D reconstruction of the device with laser radar to perform 3D reconstruction of the patient's auricle and generate a 3D model file in OBJ format. Import the file into Blender software. Based on the model file, design a gridded macroporous bracket structure with a bracket diameter of about 300μm and a channel structure diameter of about 500μm; then design a high-density and dispersed vascular bracket inside the macroporous bracket with a bracket diameter of about 500μm; save the file as an STL file for printing.

[0041] 2) Preparation of printing pre-solution: Under sterile, light-proof and 60°C heating conditions, GelMA60 freeze-dried powder was dissolved in PBS solution containing 0.25wt% LAP, and 15wt% high-concentration and 5wt% low-concentration GelMA solutions were prepared respectively. Mesenchymal stem cells of the P3 generation were cultured at a concentration of 1×10 7 The concentration of cells / mL was dispersed in a low-concentration GelMA solution to obtain a low-concentration GelMA pre-solution containing cells and a high-concentration GelMA pre-solution without cells.

[0042] 3) Preparation of the intermediate morphology of the artificial auricle with a vascularized framework: calibrate the 3D light-curing printing platform and load the STL model file. Load the low-concentration GelMA pre-solution containing cells and the high-concentration GelMA pre-solution without cells into the material tank, and use a double-nozzle double-tank structure installed in the light-curing 3D printer. The high-concentration GelMA solution prints the macroporous scaffold framework of the artificial auricle, and the low-concentration GelMA solution containing mesenchymal stem cells prints the preset vascular framework; the nozzle diameter for printing the macroporous scaffold framework is 300μm, the layer spacing is 200μm, the nozzle diameter for printing the vascular framework is 500μm, the light source is a 410nm UV light source, and the light source power is 20mW / cm 2 , the printing temperature was 37°C and the humidity was 60%. After printing, the entire scaffold was cultured in a low-glucose DMEM medium containing 10ng / ml VEGF, FGF-2, EGF and IGF-1 for 10 days, and after the angiogenic differentiation was completed, an intermediate morphology artificial auricle with a vascularized framework was obtained;

[0043] 4) Preparation of artificial auricle based on double frame: ColMA solution was prepared by dissolving ColMA in PBS containing 0.25 wt% LAP under sterile, light-proof and 60°C heating conditions, and P3 mesenchymal stem cells were cultured at 5×10 6 The density of 1000 μg / mL was dispersed in it, and then 20 ng / ml TGF-β, BMPs, IGF-1 and FGF-2 were added to the ColMA solution. After the surface of the intermediate-shaped artificial auricle was dried, the solution was completely filled into the entire auricle frame using the immersion-light curing method. After immersion for 30 seconds, a 410 nm UV light source with a light source power of 20 mW / cm 2 , irradiated for 10 seconds, repeated three times. Then, the cells were cultured in low-glucose DMEM medium containing 10 ng / ml TGF-β, BMPs, IGF-1 and FGF-2 for 6 weeks to obtain an artificial auricle material with a dual framework of vascularization and cartilage.

[0044] 5) Compared with Example 1, Example 2 uses a pre-designed artificial auricle large-pore stent frame with a larger aperture. The artificial auricle structure with a dual framework of vascularization and chondrogenesis was sliced, and the cells were found to be almost all green fluorescence under a confocal microscope using live-dead staining, accounting for up to 94%, which suggests that the cells survive well in this type of substrate; the slices were stained with toluidine blue and von Willebrand factor (vWF) immunofluorescence staining to mark the chondrocytes and vascular endothelial cells of the artificial auricle with a dual framework of vascularization and chondrogenesis respectively. It was observed under a confocal fiberscope that the number of chondrocytes uniformly marked with blue dye accounted for up to 78%, while the vascular endothelial cells formed a uniform and continuous network structure, accounting for 6%. The above phenomenon suggests that the chondrocytes in the artificial auricle with a dual framework of vascularization and chondrogenesis are well differentiated, but a large number of holes are generated, and a dense cartilage morphology cannot be presented; through the means of two-step differentiation, the vascular endothelial cells form a dense vascular network in the framework. Such a structure meets the histological structural basis for the long-term survival of the artificial auricle in the body. However, compared with Example 1, since the pore size formed is too large, the ColMA gel impregnated in the pore will degrade faster, causing the cells to detach from the auricle scaffold. The formed cartilage structure is too loose, which may lead to weak tissue mechanical properties.

[0045] Embodiment 3:

[0046] 1) Artificial auricle bracket design: Use the depth perception function in the 3D reconstruction of the device with laser radar to perform 3D reconstruction of the patient's auricle and generate a 3D model file in OBJ format. Import the file into Blender software. Based on the model file, design a gridded macroporous bracket structure with a bracket diameter of about 300μm and a channel structure diameter of about 200μm; then design a high-density and dispersed vascular bracket inside the macroporous bracket with a bracket diameter of about 500μm; save the file as an STL file for printing.

[0047] 2) Preparation of printing pre-solution: Under sterile, light-proof and 60°C heating conditions, GelMA60 freeze-dried powder was dissolved in PBS solution containing 0.25wt% LAP, and 15wt% high-concentration and 10wt% low-concentration GelMA solutions were prepared respectively. Mesenchymal stem cells of the P3 generation were cultured at a concentration of 1×10 7 The concentration of cells / mL was dispersed in a low-concentration GelMA solution to obtain a low-concentration GelMA pre-solution containing cells and a high-concentration GelMA pre-solution without cells.

[0048] 3) Preparation of the intermediate morphology of the artificial auricle with a vascularized framework: calibrate the 3D light-curing printing platform and load the STL model file. Load the low-concentration GelMA pre-solution containing cells and the high-concentration GelMA pre-solution without cells into the material tank, and use a double-nozzle double-tank structure installed in the light-curing 3D printer. The high-concentration GelMA solution prints the macroporous scaffold framework of the artificial auricle, and the low-concentration GelMA solution containing mesenchymal stem cells prints the preset vascular framework; the nozzle diameter for printing the macroporous scaffold framework is 300μm, the layer spacing is 200μm, the nozzle diameter for printing the vascular framework is 500μm, the light source is a 410nm UV light source, and the light source power is 20mW / cm 2 , the printing temperature was 37°C and the humidity was 60%. After printing, the entire scaffold was cultured in a low-glucose DMEM medium containing 10ng / ml VEGF, FGF-2, EGF and IGF-1 for 10 days, and after the angiogenic differentiation was completed, an intermediate morphology artificial auricle with a vascularized framework was obtained;

[0049] 4) Preparation of artificial auricle based on double frame: ColMA solution was prepared by dissolving ColMA in PBS containing 0.25 wt% LAP under sterile, light-proof and 60°C heating conditions, and P3 mesenchymal stem cells were cultured at 5×10 6 The density of 1000 μg / mL was dispersed in it, and then 20 ng / ml TGF-β, BMPs, IGF-1 and FGF-2 were added to the ColMA solution. After the surface of the intermediate-shaped artificial auricle was dried, the solution was completely filled into the entire auricle frame using the immersion-light curing method. After immersion for 30 seconds, a 410 nm UV light source with a light source power of 20 mW / cm 2 , irradiated for 10 seconds, repeated three times. Then, the cells were cultured in low-glucose DMEM medium containing 10 ng / ml TGF-β, BMPs, IGF-1 and FGF-2 for 6 weeks to obtain an artificial auricle material with a dual framework of vascularization and cartilage.

[0050] 5) Compared with Example 1, Example 3 uses a higher concentration of GelMA solution to print the vascular framework, with a concentration of 10wt%. The artificial auricle structure with a dual framework of vascularization and chondrogenesis was sliced, and live-dead staining was used to find that almost all cells were green fluorescent under a confocal microscope, accounting for up to 78%, which suggests that the cells survive well in this type of substrate; the slices were stained with toluidine blue and von Willebrand factor (vWF) immunofluorescence staining to mark the chondrocytes and vascular endothelial cells of the artificial auricle with a dual framework of vascularization and chondrogenesis, respectively. It was observed under a confocal fiberscope that the number of chondrocytes uniformly labeled with blue dye accounted for up to 74%, while the vascular endothelial cells formed a discontinuous network structure, accounting for 0.5%. The above phenomenon suggests that the chondrocytes in the artificial auricle with a dual framework of vascularization and chondrogenesis are evenly and densely differentiated and present a good cartilage morphology; however, since the concentration of the GelMA solution used in the preset vascular frame is too high, the efficiency of angiogenic differentiation is low, and it is difficult to form a complete and continuous vascular network structure; compared with Example 1, this embodiment may not provide a good exchange of nutrients and wastes due to the discontinuity of the vascular network structure, which may affect its long-term survival in the body.

Claims

1. A method for preparing an artificial auricle material having a composite vascularized and cartilaginized dual framework, characterized in that: The steps include: 1) Artificial auricle scaffold design: 3D modeling is performed as needed to obtain a gridded artificial auricle macroporous scaffold model, and a vascular differentiation framework is preset on it; 2) Preparation of an intermediate-form artificial auricle with a vascularized frame: Based on the multi-material multi-nozzle light-curing 3D printing technology, the artificial auricle macroporous scaffold is printed using the GelMA solution according to the model in 1), and the vascularization differentiation frame is printed in the frame using the GelMA solution containing mesenchymal stem cells (MSCs). After vascularization differentiation culture, an intermediate-form artificial auricle with a vascularized frame is obtained; 3) Preparation of artificial auricle based on dual framework: Combined with the multiple immersion-light curing method, the ColMA solution containing MSCs was introduced into the pores of the intermediate-form artificial auricle, and after chondrogenic differentiation culture, an artificial auricle material with both vascularized and chondrogenic dual frameworks was obtained.

2. The method for preparing an artificial auricle material having a composite vascularized and cartilaginized dual framework according to claim 1, characterized in that: In step 1), the mesh scaffold diameter of the meshed artificial auricle macroporous scaffold model is 300-500 μm, and the pore structure diameter is 200-300 μm; the preset angiogenic differentiation framework is angiogenic scaffolds dispersed in the macroporous scaffold, and the scaffold diameter is 500-700 μm.

3. The method for preparing an artificial auricle material having a composite vascularized and cartilaginized dual framework according to claim 1, characterized in that: In step 2), the concentration of the GelMA solution for printing the artificial auricle pore scaffold is 15-20wt%, and the dissolution temperature is 60-80°C; the concentration of the GelMA solution carrying mesenchymal stem cells is 5-7.5wt%, and the dissolution temperature is 60-80°C, and it is cooled to 35-37°C after complete dissolution; a photoinitiator is added to the above two GelMA solutions, which is one or more of LAP, Irgacure, and I2959, with a concentration of 0.1-0.25wt%, and the dissolution is carried out under light-proof conditions; the MSCs are within the P2-P5 generation, with a concentration of 1×10 7 The concentration of 100 / mL was dispersed in the GelMA solution.

4. The method for preparing an artificial auricle material having a composite vascularized and cartilaginized dual framework according to claim 1, characterized in that: In step 2), the multi-material multi-nozzle light-curing 3D printer uses a dual-nozzle dual-tank structure, the light source is a 350-410nm UV light source, and the light source power is 20-40mW / cm 2 , the nozzle diameter for printing the artificial auricle pore scaffold is 300-500μm, the printing layer thickness is 300-500μm, and the layer interval is 200-300μm; The nozzle for printing the angiogenic differentiation framework is 500-700 μm, the printing layer thickness is 500-700 μm; the printing temperature is 35°C-37°C, and the humidity is 60%-80%.

5. The method for preparing an artificial auricle material having a composite vascularized and cartilaginized dual framework according to claim 1, characterized in that: In step 2), the medium for the angiogenic differentiation culture uses a basal medium of one or more of low-glucose DMEM or α-MEM, and the added angiogenic factors are one or more of VEGF, FGF-2, EGF and IGF-1, with a concentration range of 5-10 ng / ml.

6. The method for preparing an artificial auricle material having a composite vascularized and cartilaginized dual framework according to claim 1, characterized in that: In step 3), the ColMA solution has a concentration of 5-7.5wt%, a dissolution temperature of 60-80°C, and is cooled to 35-37°C after complete dissolution. The photoinitiator added is one or more of LAP, Irgacure, and I2959, with a concentration of 0.1-0.25wt%; the MSCs generation is P2-P5, and the concentration is 5×10 6 Pieces / ml.

7. The method for preparing an artificial auricle material having a composite vascularized and cartilaginized dual framework according to claim 1, characterized in that: In step 3), the basal medium for chondrogenic differentiation culture is one or more of low-glucose DMEM or α-MEM; the chondrogenic factors added in the ColMA solution and the culture medium mainly include the following: one or more of TGF-β, BMPs, IGF-1 and FGF-2, the concentration of the chondrogenic factors in the ColMA solution is 10-20 ng / ml, and the concentration of the chondrogenic factors in the culture medium is 5-10 ng / ml.

8. The method for preparing an artificial auricle material having a composite vascularized and cartilaginized dual framework according to claim 1, characterized in that: In step 3), the immersion time of the cyclic immersion-light curing method is 30-50 seconds, and the light source during light curing is a 350-410nm UV light source with a power of 20-40mW / cm 2 , the irradiation time is 10-15s, and the number of cycles is 3-5 times.

9. An artificial auricle material having a composite vascularized and cartilaginized dual framework, characterized in that: The method is prepared by the method according to any one of claims 1 to 8.

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