Auricular cartilage scaffold material as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological materials, and particularly relates to an auricular cartilage scaffold material as well as a preparation method and application thereof. According to the preparation method of the auricular cartilage scaffold material, thermoplastic polyurethane is selected as a base material, sodium chloride is taken as a pore-forming agent, and the auricular scaffold material matched with the mechanical property of the auricular cartilage of a normal person is successfully constructed by regulating and controlling material processing parameters. Experiments prove that the scaffold material prepared by the invention not only has structural stability and biocompatibility, but also has mechanical properties matched with those of natural auricular cartilage. Therefore, a new theoretical basis and a technical strategy can be provided for personalized treatment of auricle reconstruction, and a solid foundation can be laid for clinical transformation and application of a novel auricle support.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomaterials, and particularly relates to an auricular cartilage scaffold material, a preparation method thereof, and an application thereof. Background Art

[0002] Congenital middle and outer ear malformations are birth defect diseases, and the main phenotypes are microtia, external auditory canal stenosis or atresia, and middle ear malformations. The clinical manifestations are appearance deformities and hearing impairments. Among them, microtia shows partial or complete auricular defects, ranking among the top 10 in more than 8,000 birth defect diseases in China. At the same time, acquired factors such as auricular trauma and after-tumor surgery also lead to a large number of cases of auricular defects. The auricular defect deformity will cause serious psychological disorders in patients, thereby affecting learning and life.

[0003] At present, the main treatment method for microtia (auricular defect) is auricular reconstruction, and its core technology lies in the auricular cartilage scaffold. At present, autologous costal cartilage scaffolds (93.3%) and artificial material Medpor scaffolds (6.3%) are mainly used at home and abroad. Autologous costal cartilage is widely used because of its good compatibility, toughness, easy carving, and good psychological feelings of patients. However, there are still problems such as donor site injury (pain, scar, deformity), possible complications (hemopneumothorax), the effect completely depending on the surgeon's technique, the operation time (age) being limited due to the need for a certain amount and toughness, and the operation time for carving the scaffold being significantly prolonged. Although the Medpor scaffold has a lifelike shape and good tissue compatibility, due to its high hardness, no elasticity, and easy surface skin rupture and infection after compression, it is difficult to maintain for life, resulting in limited application.

[0004] Tissue engineering bionic auricular cartilage scaffolds incorporate all the above advantages and avoid all the disadvantages. However, since the scaffolds in tissue engineering are degradable materials, their degradation rate is faster than the regeneration rate of implanted chondrocytes, and the mechanical strength decays too fast, making it difficult to meet the long-term support requirements of the auricle, resulting in the inability to maintain the shape of the reconstructed auricle and thus not being able to be applied clinically.

[0005] In summary, developing a new type of auricular cartilage scaffold with mechanical properties adapted to those of normal human auricular cartilage has become the key to breaking through the current clinical bottleneck and can also provide a theoretical basis and technical strategy for personalized auricular reconstruction. Summary of the Invention

[0006] To overcome the deficiencies in the prior art, the object of the present invention is to provide an auricular cartilage scaffold material, its preparation method and application. The preparation method of the auricular cartilage scaffold material provided by the present invention uses existing thermoplastic polyurethane, and only adjusts the pore size, hardness and elasticity by mixing with sodium chloride, so that the mechanical properties of the prepared auricular cartilage scaffold material are highly compatible with those of normal human auricular cartilage, without changing its chemical structure and other physical properties. After implantation, the scaffold material can effectively achieve interface integration with the host tissue without cell culture, thus providing a safe, efficient and widely applicable solution for clinical auricular reconstruction surgery.

[0007] To achieve the above object, in the first aspect of the present invention, a preparation method of an auricular cartilage scaffold material is provided, including the following steps: Mix thermoplastic polyurethane and sodium chloride evenly to obtain a mixture; inject the mixture into an auricular mold, perform low-temperature curing treatment, and then demold to obtain a formed material; wash the formed material to remove sodium chloride to obtain the auricular cartilage scaffold material; in the mixture, the mass percentage of sodium chloride is 25% - 75%.

[0008] As a preferred scheme, the step of mixing thermoplastic polyurethane and sodium chloride evenly is as follows: heat the thermoplastic polyurethane to melt it to reach the viscous flow state, and then add sodium chloride and stir to mix evenly.

[0009] As a preferred scheme, the temperature of the heating and melting is 170 - 190 °C.

[0010] As a further preferred scheme, the temperature of the heating and melting is 180 °C.

[0011] As a further preferred scheme, in the mixture, the mass percentage of sodium chloride is 50%.

[0012] As a preferred scheme, the structure of the auricular mold matches the auricular structure of the application object.

[0013] The present invention does not specifically limit the preparation process of the auricular mold. Those skilled in the art can adopt well-known preparation methods in the field, such as manual production or 3D printing technology, etc. It only needs to ensure that the structure of the auricular mold matches the auricular structure of the application object, such as the human auricular structure, so as to achieve the purpose of truly simulating the appearance of human auricular cartilage.

[0014] As a preferred scheme, the temperature of the low-temperature curing treatment is -10 - -30 °C, and the time of the low-temperature curing treatment is 10 - 30 h.

[0015] As a preferred scheme, the solvent used for washing is water, and the washing time is 30 - 100 h; after the washing, it also includes drying and equilibration steps.

[0016] The second aspect of the present invention provides an auricular cartilage scaffold material prepared by the above preparation method.

[0017] The third aspect of the present invention provides the application of the above auricular cartilage scaffold material in the preparation of a human auricular cartilage scaffold.

[0018] The technical solution of the present invention has the following advantages and beneficial effects: The preparation method of the auricular cartilage scaffold material provided by the present invention is based on the long-existing problem of mismatched mechanical properties of scaffold materials in the field of auricular reconstruction for auricular deformities. First, the Young's elastic modulus of human auricular cartilage and the hardness data of ex vivo auricular cartilage are measured. At the same time, thermoplastic polyurethane is selected as the substrate and sodium chloride is used as the pore-forming agent. By adjusting the material processing parameters and combining three-dimensional scanning and model design, an auricular cartilage scaffold material highly compatible with the mechanical properties of normal human auricular cartilage is successfully constructed.

[0019] Comprehensive experiments have confirmed that the scaffold material prepared by the present invention not only has both structural stability and biocompatibility, especially its mechanical properties are matched with those of natural auricular cartilage. Therefore, it can be used as an ideal alternative material for existing cartilage scaffolds. In particular, the reported auricular cartilage scaffolds often need to be transplanted after autologous cell growth, while the auricular cartilage scaffold material prepared by the present invention does not require cell culture and can be directly transplanted, saving the processes of cell separation, amplification and re-implantation, and greatly improving the preparation efficiency of the scaffold material.

[0020] Therefore, the present invention can provide a new theoretical basis and technical strategy for the personalized treatment of auricular reconstruction, and also lay a solid foundation for the clinical transformation and application of new auricular scaffolds. Description of the Drawings

[0021] Figure 1 It is the test result of the elasticity of the auricular cartilage of the subject in the present invention; Figure 2 It is the test result of the hardness of the auricular cartilage of the subject in the present invention; Figure 3 It is the test result of the thermogravimetric curve of thermoplastic polyurethane and the loss factor of the storage modulus in the present invention; Figure 4 It is the scanning electron micrograph of the thermoplastic polyurethane material with different sodium chloride dosages in the present invention; Figure 5 It is the test result of the compressive stress-strain curve, compressive modulus and energy dissipation rate of polyurethane with different sodium chloride dosages in the present invention; Figure 6 It is the result of the change of strain with time under 5 MPa compression of polyurethane with different sodium chloride dosages in the present invention; Figure 7 Shear wave elastography images of polyurethanes with different sodium chloride dosages in the present invention; Figure 8 Molding drawings of the human ear scanning model and auricular cartilage in the present invention; Figure 9 Process flow chart for preparing the auricular cartilage scaffold material in the present invention; Figure 10 Thermogravimetric analysis, compression test, and creep test results of the polyurethane material before processing and the processed polyurethane auricular cartilage scaffold material in the present invention; Figure 11 HE staining and Masson staining results of the scaffold-tissue interface samples in the present invention; Figure 12 Thermogravimetric analysis, compression test, and creep test results of the scaffold material taken out after the animal experiment in the present invention. Specific embodiments

[0022] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be described clearly and completely below in conjunction with specific embodiments. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0023] In the following embodiments of the present invention, the thermoplastic polyurethane used is a commercially available product (TPU, grade RxT90A) from Covestro Polymer Co., Ltd., which belongs to medical polyurethane. Sodium chloride (NaCl) is 10-80 mesh and has a purity of more than 99.5%, and is a commercially available product from Macklin Reagent Co., Ltd. Other raw materials not specified are all conventional materials that can be obtained through commercial channels in the art. Example 1

[0024] This example provides an auricular cartilage scaffold material, and its preparation method includes the following steps: The thermoplastic polyurethane was placed in an oil bath at 180 °C and heated to the molten state until it was completely melted and reached the viscous flow state. Then, sodium chloride with a mass ratio of 50% was added and stirred until homogeneous to obtain the TPU-NaCl mixture. Among them, the dosage of sodium chloride was 50% of the mass of the TPU-NaCl mixture. The TPU-NaCl mixture was injected into a pre-prepared auricle mold (the structure of the auricle mold matched the structure of the human auricle), and then the mold was placed in a low-temperature environment at -20 °C for curing treatment for 24 h. Subsequently, demolding was carried out to obtain a preliminarily formed scaffold material. The preliminarily formed scaffold material was soaked in deionized water for washing, and the deionized water was changed every 12 h, and continuous washing was carried out for 48 h. After washing was completed, the scaffold material was dried in an oven at 60 °C to remove excess moisture. Then, the dried scaffold material was soaked in deionized water again until the material reached an equilibrium state with water, that is, the auricular cartilage scaffold material of this example was obtained.

[0025] Experimental Example 1: Determination of the elasticity and hardness of auricular cartilage 1.1. Elasticity test of auricular cartilage Test subjects: Forty (80 ears) healthy volunteers were included as subjects, with a male-to-female ratio of 1:1 and an average age of 32.93 ± 15.72 years. Inclusion criteria: ① No history of ear surgery; ② No history of systemic diseases; ③ The auricular anatomical structure is intact. Exclusion criteria: ① Congenital ear malformations (such as microtia); ② Traumatic auricular defects; ③ Autoimmune chondropathy (such as relapsing polychondritis); ④ Severe systemic diseases.

[0026] Test method: The Aixplorer shear wave ultrasonic elastography instrument was used to measure the elasticity of the auricular cartilage of the subjects, and 10 repeated measurements were performed on each auricle of the subjects. When processing the data, abnormal data points with a coefficient of variation exceeding 15% were excluded, and then the remaining valid data were calculated by arithmetic mean.

[0027] The test results of the elasticity of auricular cartilage are as Figure 1 shown. Figure 1 Among them, from left to right are: the relationship between age and the Young's elastic modulus of auricular cartilage, the relationship between BMI and the Young's elastic modulus of auricular cartilage, and the relationship between gender and the Young's elastic modulus of auricular cartilage.

[0028] It can be Figure 1 seen that the average Young's elastic modulus of auricular cartilage measured by shear wave elastography technology is 112.93 ± 5.04 kPa. Figure 1A scatter plot combined with correlation analysis showed no significant correlation between age (r=-0.018, p>0.05) or BMI (r=0.054, p>0.05) and elasticity. Gender difference analysis showed that the average Young's modulus for men was 114.16±5.43 kPa, slightly higher than that for women (111.70±4.34 kPa), but the distribution characteristics of the two data sets were generally consistent.

[0029] 1.2. Auricular cartilage hardness test Participants: Forty patients (40 ears) diagnosed with otitis media / cholesteatoma undergoing surgical treatment were enrolled. The male-to-female ratio was 0.43:1, and the mean age was 52.85±15.20 years. Auricular cartilage samples were obtained during surgery. Inclusion criteria: ① Confirmed cholesteatoma or chronic suppurative otitis media by temporal bone CT scan; ② Intraoperative auricular cartilage sampling for middle ear reconstruction was required. Exclusion criteria: ① Congenital ear malformation (e.g., microtia); ② Traumatic auricular defect; ③ Autoimmune chondropathy (e.g., relapsing polychondritis); ④ Severe systemic disease.

[0030] Test Method: Auricular cartilage specimens obtained during surgery were immediately placed in a sterile bag for testing. The specimen was then placed between the spherical indenter and circular platform of a Shore durometer. The screw was pulled downward at a continuous, steady speed, and the upper limit value, which did not change significantly within 3 seconds, was read. All procedures were performed by the same professional, with three repeated measurements of the target area taken, and the average result was calculated.

[0031] The test results of the auricular cartilage hardness are as follows Figure 2 shown. Figure 2 In the figure, from left to right: the relationship between age and auricular cartilage hardness, the relationship between BMI and auricular cartilage hardness; the relationship between gender and auricular cartilage hardness.

[0032] Depend on Figure 2 It can be seen that the average Shore hardness of the auricle cartilage samples measured by a hardness tester is 72.73±5.04C. Figure 2 A scatter plot combined with correlation analysis revealed a moderate negative monotonic correlation between age (r=-0.4486, p<0.05) and cartilage stiffness. There was no significant linear correlation between BMI (r=0.0658, p>0.05) and cartilage stiffness. Gender differences revealed that the average auricular cartilage stiffness in men was slightly higher at 75.20±5.91°C compared to 71.24±3.84°C in women, suggesting a correlation with sex hormone regulation of cartilage matrix metabolism. However, the distribution characteristics of the two data sets were generally consistent.

[0033] In the above experiments, through the mechanical property tests of auricular cartilage by in vivo and ex vivo measurement methods, the mechanical property characteristics of human auricular cartilage were preliminarily revealed, providing a theoretical basis for constructing an auricular cartilage scaffold adapted to the mechanical properties of normal human auricular cartilage. On the one hand, in order to ensure that the measurement results can accurately reflect the true mechanical properties of normal human auricular cartilage in vivo, the present invention uses shear wave elastography (SWE) to carry out the evaluation of the mechanical properties of in vivo auricular cartilage, so as to obtain the elastic modulus of auricular cartilage. On the other hand, the present invention immediately conducts tests after taking ex vivo auricular cartilage samples, effectively avoiding the influence of tissue activity attenuation on the measurement results. The results show that the average Young's elastic modulus of auricular cartilage is 112.93 ± 5.04 kPa, and the average hardness of auricular cartilage is 72.73 ± 5.04 C. The relationship between age, BMI, gender and hardness is consistent with the trend of the results measured by shear wave elastography. Therefore, the present invention selects the mean value of the hardness test results for material matching. In this process, through the rigorous process of first matching with the hardness mean value and then verifying with shear wave elastography, the present invention can minimize errors and comprehensively improve the credibility of the data results.

[0034] Experimental Example 2: Construction and Preparation of Auricular Cartilage Scaffold Materials 2.1 Determination of Processing Conditions for Thermoplastic Polyurethane Materials Place the thermoplastic polyurethane (hereinafter referred to as TPU) raw material in a thermogravimetric analyzer and heat it from 50°C to 600°C at a heating rate of 10°C / min. By analyzing the curve of the change in TPU mass with temperature, determine its thermal decomposition temperature. At the same time, conduct dynamic thermal analysis on TPU in the temperature range of -200°C to 200°C to study the variation laws of its storage modulus (E') and loss factor (tanδ) with temperature. Further, by analyzing the peak position of the tanδ curve, determine the glass transition temperature (Tg) of TPU. This temperature is the critical point for the material to transform from the glassy state to the rubbery state, and it has important guiding significance for temperature control during the processing. The thermogravimetric curve of thermoplastic polyurethane and the measurement results of the loss factor of the storage modulus are as Figure 3 shown. Among them, Figure 3 a is the thermogravimetric curve of thermoplastic polyurethane; b is the loss factor of the storage modulus of thermoplastic polyurethane.

[0035] From Figure 3It can be seen that the thermogravimetric curve (TG) of thermoplastic polyurethane shows that the material starts to decompose at 267.8 °C until complete decomposition. This decomposition temperature range conforms to the typical thermal decomposition characteristics of polyurethane, indicating that the material has good thermal stability at temperatures below 267.8 °C (Figure a). When performing dynamic thermal analysis tests within the stable temperature range of polyurethane, the storage modulus (E') starts to decrease significantly at 124.7 °C. This indicates that the material begins to transform from the high elastic state to the viscous flow state near this temperature. Further tests reveal that at 180 °C, the loss factor (tanδ) of polyurethane reaches 0.3, and at this time the material is already in the viscous flow state (Figure b). At 180 °C, the loss factor of polyurethane is relatively high, indicating that the internal friction of the material is large, the polyurethane is in the viscous flow state, and has good fluidity and processability. This temperature is much lower than the thermal decomposition temperature of the material, ensuring that thermal degradation does not occur during the processing. Based on the above test results, the processing temperature of thermoplastic polyurethane is determined to be 180 °C.

[0036] 2.2. Pore regulation and mechanical property testing of thermoplastic polyurethane materials The TPU material is placed in an oil bath at 180 °C and heated to the molten state until it melts and reaches the viscous flow state. After the TPU melts, sodium chloride (NaCl) with different weight ratios (the mass ratios of NaCl in the TPU-NaCl mixture are 0%, 25%, 50%, and 75% respectively) is added and stirred at high speed until the mixture is homogeneous, obtaining the TPU-NaCl mixture. NaCl is used as a pore inducer, and its addition amount directly affects the porosity and pore size distribution of the TPU material. During the stirring process, it is necessary to ensure the homogeneity of the mixture to avoid non-uniform pore distribution. The well-stirred TPU-NaCl mixture is quickly injected into a preheated mold to ensure that the mixture is evenly distributed in the mold. Subsequently, the mold is placed in a low-temperature environment at -20 °C for 24 h. The purpose of the low-temperature treatment is to quickly cool and solidify the mixture, and at the same time prevent the recrystallization or migration of NaCl during the cooling process, thereby ensuring the stability of the pore structure. After the low-temperature treatment, the NaCl particles are removed by dissolution to form a uniformly distributed pore structure, obtaining TPU materials with different porosities.

[0037] The above-prepared TPU materials with different porosities are processed into cylinders with a diameter of 20 mm and a height of 8 mm. Compression tests are performed on the prepared TPU cylinders to obtain the compression stress-strain curves. During the test, the maximum strain is controlled at 50% to ensure that the material does not break within the test range. The compression test results can reflect performance parameters such as the elastic modulus and energy dissipation rate of the material.

[0038] Furthermore, at a fixed strain of 5 MPa, TPU materials with different porosities were compressed for a duration of 3600 s. By observing the deformation of the materials under long-term compression, their creep behavior can be evaluated. The creep test results can reflect the stability and reliability of the materials under long-term use conditions.

[0039] Furthermore, TPU materials with different porosities were processed into cuboids with dimensions of 10×10×50 mm. The prepared TPU cuboids were measured using shear wave elastography, and the measurement method was the same as in Test Example 1. Then, the final processed porosity was determined based on the average Young's elastic modulus of normal human auricular cartilage.

[0040] In this experiment, the scanning electron microscopy (SEM) images of thermoplastic polyurethane materials with different sodium chloride dosages are as Figure 4 shown. As Figure 4 can be seen, through the NaCl porogen method, the porosity of the polyurethane scaffolds can be effectively regulated. As the NaCl dosage increases, the porosity of the polyurethane scaffolds shows an upward trend. When the NaCl addition amount is 25 wt.%, only a small number of pores are observed in the polyurethane scaffolds, and the pore distribution is relatively sparse; while when the NaCl addition amount increases to 75 wt.%, the interior of the polyurethane scaffolds is almost completely occupied by pores, presenting a highly porous structure.

[0041] The measurement results of the compression stress-strain curves, compression moduli, and energy dissipation rates of polyurethane materials with different porosities are as Figure 5 shown. Figure 5 In Figure 5 it, a is the compression stress-strain curve; b is the compression modulus; c is the energy dissipation rate. As

[0042] can be seen, the polyurethane without pores exhibits obvious linear elasticity, with a compression modulus as high as 23.75 MPa. The material has high elasticity and a relatively hard texture. This high modulus property makes it perform well in applications requiring high rigidity and strength, but its energy dissipation ability is relatively weak. As the porosity increases, the compression modulus of the polyurethane decreases significantly. When the sodium chloride dosage increases to 50 wt.%, the elastic modulus of the polyurethane scaffold drops to approximately 11.24 MPa. At this time, the texture of the material becomes soft. The increase in porosity not only reduces the elastic modulus but also significantly improves the energy dissipation rate of the polyurethane. For example, the energy dissipation rate of the polyurethane scaffold with a 50 wt.% sodium chloride dosage can reach 56.37%. This high energy dissipation ability enables the material to better absorb and disperse energy when subjected to external forces.

[0042] Creep refers to the phenomenon that materials gradually deform under long-term stress. The results of the strain change of polyurethane with different porosities under 5 MPa compression over time are as Figure 6 shown. Figure 6The experimental results show that when the sodium chloride dosage increases to 75 wt.%, the structural integrity of the polyurethane scaffold is greatly affected, resulting in it being more prone to creep under long-term loading. In contrast, polyurethane scaffolds with lower porosities (such as sodium chloride dosages of 25 wt.% or 50 wt.%) exhibit better creep resistance under the same stress conditions. The polyurethane scaffolds can still maintain stable mechanical properties under long-term loading, indicating that their internal structure can disperse stress to a certain extent, thereby reducing the occurrence of creep.

[0043] Shear wave elastography images of polyurethane materials with different sodium chloride dosages are shown as Figure 7 follows. Figure 7 The results show that when the sodium chloride dosage is 50 wt.%, the porosity of the polyurethane is close to the average value of shear wave elastography of normal human auricular cartilage. Based on this, the target sodium chloride dosage for the auricular cartilage scaffold is finally set at 50 wt.%.

[0044] 2.3. Construction and manufacturing of auricular scaffold model In this experiment, 3D technology is used to construct the auricular scaffold model. A handheld 3D scanner is used to perform high-precision scanning of the human ear to obtain the three-dimensional point cloud data of the human ear. After the scanning is completed, a three-dimensional digital model of the human ear is obtained. The scanned human ear model (as shown in Figure 8 a) is imported into Geomagic Wrap software for model trimming. By removing scanning noise, filling holes, and smoothing the surface, the geometric accuracy and surface quality of the model are optimized. The trimmed model can accurately reflect the true shape of the human ear. The optimized human ear model is imported into SolidWorks software for mold design, and a mold for TPU auricular cartilage implantation processing is prepared according to the geometric characteristics of the human ear model.

[0045] In mold preparation, special attention is paid to the regions of the auricular cartilage helix, antihelix, triangular fossa, scaphoid fossa, and concha cavity. The shapes and structures of these parts are crucial for the overall appearance display of the auricle. The specific mold opening strategy is: opening the mold along the helix region on the front of the auricular cartilage (as shown in Figure 8 b). This method can ensure that the auricular cartilage scaffold is not damaged during the demolding process while retaining the key structural features of the auricle.

[0046] Furthermore, the processing of the TPU auricular cartilage scaffold is the same as the optimal processing method of TPU in Example 1. The TPU-NaCl mixture (melting temperature 180 °C, mass ratio of sodium chloride 50%) is injected into the mold, and then cryo-treated for 24 hours. After the cryo-treatment is completed, the mold is demolded to remove the preliminarily formed TPU auricular cartilage scaffold. The formed auricular cartilage scaffold is soaked in deionized water for washing, and the deionized water is changed every 12 hours for continuous washing for 48 hours. This process is to remove the residual NaCl particles inside the scaffold to ensure the cleanliness and uniformity of the pore structure. After washing, the scaffold is dried in an oven at 60 °C to remove excess moisture. The dried TPU auricular cartilage scaffold is soaked in deionized water again until the material reaches an equilibrium state with the water. This step is used to simulate the water absorption behavior of the scaffold in the physiological environment to ensure its stability and biocompatibility in actual applications. After the water equilibrium treatment, a polyurethane artificial auricle scaffold that can be used for subsequent research or clinical applications is finally obtained.

[0047] The process flow chart of the above-mentioned polyurethane artificial auricle scaffold, that is, the auricular cartilage scaffold material of the present invention, is as Figure 9 shown. Figure 9 In it, a is the prepared auricle scaffold mold; b is the high-temperature melting of the thermoplastic polyurethane material; c is the prepared polyurethane artificial auricle scaffold. Figure 9 In it, the uniformly stirred TPU-NaCl mixture is quickly injected into the pre-prepared auricle mold. After the mixture cools and solidifies in the mold, the demolding operation is carried out. The demolded polyurethane auricle scaffold needs to be further post-treated to remove NaCl particles. The prepared polyurethane auricle scaffold can simulate the mechanical properties and appearance of the human auricular cartilage and is suitable for auricle reconstruction.

[0048] Furthermore, according to the method described above, thermogravimetric analysis, compression test, and creep test are carried out on the polyurethane material before processing and the polyurethane auricular cartilage scaffold after processing, and the test results are as Figure 10 shown. Figure 10 In it, a is the thermogravimetric curve; b is the compression stress-strain curve; c is the change of strain with time under 5 MPa compression.

[0049] From Figure 10It can be seen that the thermogravimetric curve of the material shows no obvious change, which is consistent with the unprocessed polyurethane material (Figure a). This indicates that the temperature and process conditions adopted during the processing do not damage the chemical structure of the polyurethane, and the thermal stability of the material is maintained. The results of the mechanical property tests show that the processed auricular cartilage scaffold is consistent with the unprocessed polyurethane material in terms of key mechanical indexes such as elastic modulus and compressive strength (Figure b). This shows that the processing process (including NaCl pore formation and mold forming) does not have a negative impact on the mechanical properties of the polyurethane, and the mechanical characteristics of the material are retained after processing. The results of the creep test show that there is no obvious creep phenomenon in the processed polyurethane auricular cartilage scaffold under long-term stress conditions (Figure c). This indicates that the processing process does not damage the internal structure of the polyurethane, and the material can still maintain good mechanical stability during long-term use.

[0050] Experimental Example 3: In-vivo Experimental Verification of the Auricular Scaffold Material The present invention has confirmed that the combination of the NaCl pore formation method and the mold perfusion molding technology shows unique advantages in constructing a three-dimensional porous structure and simulating the mechanical properties of normal human auricular cartilage. However, the mechanical stability, tissue integration ability, biocompatibility, anti-infection ability, etc. of the scaffold material after implantation in the body still need to be verified through in-vivo experiments. In this experiment, nude mice were used as the experimental model to conduct in-vivo implantation experiments, observe the appearance changes of the scaffold after implantation, and evaluate the mechanical properties and in-vivo biocompatibility of the scaffold through a histological and biomechanical detection system, providing key experimental basis for clinical transformation.

[0051] 3.1 Animal Selection and Implantation of the Auricular Scaffold Animal Selection: Subcutaneous implantation studies of the TPU auricular scaffold prepared in the same batch were carried out on rabbits, rats and nude mice. Through preliminary comparative experiments, the present invention selected immunodeficient nude mice as the experimental animal model. This model not only has anatomical characteristics such as thin skin and a thin subcutaneous fat layer, but also lacks T cell-mediated immune rejection reactions, can effectively simulate the clinical scenario of allogeneic scaffold implantation, and at the same time meets the needs of long-term dynamic observation. During the formal experiment: CD-1 nude mice (n = 6), female, 6 weeks old, 25 - 30 g were taken. They were raised under SPF environmental levels, with an environmental temperature of 23°C, an environmental humidity of 39.5 - 68.9%, lighting: automatic lighting, 12 hours of light and dark alternation. The air cleanliness was grade 5, and all drinking water and feed were disinfected.

[0052] Scaffold implantation: Six auricular cartilage scaffolds of the same batch prepared under the optimal parameters of Example 2 were sterilized with ethylene oxide and placed in a sterile container for standby. All surgical instruments were sterilized by high-pressure steam for standby. Six-week-old CD-1 nude mice were placed in a laminar flow hood, and a special inhalation anesthetic machine for small animals was connected (oxygen flow rate was set at 2 L / min). Isoflurane inhalation anesthesia was used: the initial induction anesthesia concentration was 2.5% (volume fraction). After the animal's corneal reflex disappeared and muscle relaxation occurred (about 2 - 3 min), the maintenance anesthesia concentration was adjusted to 1.5 - 2.0%. The skin of the surgical area was disinfected three times with povidone-iodine solution, and the diameter of the disinfected area was ≥5 cm. A skin incision about 1.5 - 2.0 cm long was made longitudinally along the back, and blunt dissection was performed between the subcutaneous fascia layer and the dermis layer to form a pocket-like cavity. Care was taken to avoid damaging the subcutaneous vascular network during the dissection. The TPU auricular scaffold in the sterile container was implanted into the dissected cavity (the auricular scaffold did not require cell culture and was directly transplanted). A drainage tube was placed at the lowest position of the cavity, and the subcutaneous tissue and skin were sutured intermittently with absorbable sutures to ensure that the drainage tube was fixed in place. The skin of the incision and the surrounding area was disinfected again with povidone-iodine solution. After the animal fully recovered from anesthesia, it was returned to the SPF-class animal room for routine feeding.

[0053] 3.2 In vivo test Postoperative observation and mechanical property test: The status of the experimental animals was recorded daily from the 1st day to the 1st month after surgery, including mental state, food and water intake, incision healing process (presence of redness / swelling / effusion / crusting), and appearance changes at the scaffold implantation site. Key monitoring was carried out for the occurrence of complications such as signs of infection and scaffold exposure. On the 3rd day after surgery, the area around the drainage tube was disinfected with povidone-iodine, the skin sutures were removed, and the drainage tube was removed. The characteristics of the drainage fluid were observed and the drainage volume was recorded. At 1 month after surgery, the experimental animals were sacrificed by an overdose of isoflurane inhalation anesthesia combined with cervical dislocation. The newly formed tissue around the scaffold was completely dissected, and the scaffold-tissue interface samples were obtained. The scaffold-tissue interface samples were immediately placed in 10% neutral formalin fixative and stored in a constant temperature refrigerator at 4°C for HE staining and Masson staining analysis. Then, the TPU cartilage scaffold material was carefully dissected and separated, and the TPU scaffold was cut into standard cylinders with a diameter of 20 mm and a height of 8 mm using a precision wire saw for mechanical property characterization.

[0054] Among them, the processes of HE staining and Masson staining are as follows: Take a fresh tissue sample (volume 1×1×1 cm³), immediately place it in a tissue embedding cassette containing 4% paraformaldehyde (pH 7.4), and fix it at 4°C for 24 hours. Rinse the fixed tissue with 0.9% normal saline under running water for 2 hours. Immerse it successively in gradient ethanol solutions (70%, 80%, 90%, 95%Ⅰ, 95%Ⅱ, 100%Ⅰ, 100%Ⅱ), with each level treated at room temperature for 1 hour and the fresh solution replaced at each level. Immerse the tissue successively in xyleneⅠand xyleneⅡfor 20 minutes each, operating at room temperature. Place the cleared tissue in an incubator at 60°C and impregnate it successively with paraffinⅠ(1 h), paraffinⅡ(1.5 h), and paraffinⅢ(1.5 h), replacing the fresh paraffin at each level. Use a tissue embedding machine (Kedi KD-BM) for directional embedding, with the embedding temperature controlled at 62±1°C. Immediately place it on an ice table at -20°C for rapid cooling after embedding, and separate the wax block after the embedding bottom mold solidifies. Use a cryostat (Leica CM1850), adjust the knife holder angle to -5° to -10° after fixing the wax block, and cut 4-5 μm continuous sections. Place the sections in distilled water at 42-45°C to flatten them, and pick up the sections with the tissue side down using a glass slide pretreated with APES. Bake in an incubator at 37°C for 16 h to enhance the adhesion between the tissue and the slide. Then perform HE staining and Masson staining on the prepared tissue sections, and finally conduct microscopic examination.

[0055] The HE staining and Masson staining results of the scaffold-tissue interface samples are as Figure 11 shown. Figure 11 follows. In the figure, a is the HE staining result; b is the Masson staining result. Figure 11In this study, histological observations were carried out on the scaffold-tissue interface samples. The results showed that the skin tissue structure presented basically normal morphological characteristics. Its structure was composed of three layers in an orderly manner: the epidermis, the dermis, and the subcutaneous tissue. Various accessory organs could be clearly observed (Figure a). The epidermis showed a typical five-layer structure, from the deep layer to the superficial layer, namely the basal layer, the spinous layer, the granular layer, the clear layer, and the stratum corneum. The dermis was composed of the papillary layer in the superficial layer and the reticular layer in the deep layer, belonging to irregular dense connective tissue. Its main components included fibers (including collagen fibers, reticular fibers, and elastic fibers), matrix, and cell components. Among them, the fiber components dominated, and a small amount of matrix and cell components were distributed between the fibers (Figure b). The subcutaneous tissue was located below the dermis and was connected to tissues such as the muscular fascia below. It was composed of loose connective tissue and fat lobules and was also called the subcutaneous fat layer. It contained blood vessels, lymphatic vessels, nerves, eccrine sweat glands, and apocrine sweat glands. Accessory organs included hair, sebaceous glands, sweat glands, etc. The main purpose of Masson staining was to clearly distinguish collagen fibers and muscle fibers in the tissue. After Masson staining, muscle fibers showed distinct red, while collagen fibers showed blue. This staining effect provided a clear visual basis for in-depth analysis of the tissue microstructure.

[0056] Furthermore, the mechanical property tests were carried out by referring to the aforementioned method. Thermogravimetric analysis, compression tests, and creep tests were performed on cylinders with a diameter of 20 mm and a height of 8 mm prepared from the scaffold material. The results of the thermogravimetric analysis, compression tests, and creep tests obtained from the tests are as Figure 12 shown. Figure 12 In this figure, a is the thermogravimetric curve of the polyurethane cartilage scaffold after removal; b is the compression stress-strain curve of the polyurethane cartilage scaffold after removal; c is the change of strain with time under 5 MPa compression of the polyurethane cartilage scaffold after removal.

[0057] After the animal experiment was completed, the polyurethane scaffold was taken out and the mechanical properties of the material were characterized again. Figure 12 The results showed that there were no obvious changes in the mechanical properties of the scaffold before and after implantation. The thermogravimetric curve determined that no new weight loss regions appeared in the material, and the decomposition temperature remained unchanged compared with that before implantation (Figure a). This indicated that the polyurethane did not decompose or denature. The mechanical tests showed that the mechanical properties of the material were still maintained after in vivo implantation, which verified that the properties of the material did not degenerate (Figure b). The creep test results showed that in vivo implantation did not affect the mechanical stability of the material (Figure c).

[0058] Based on the above results, it can be seen that the primary challenge faced by existing auricle reconstruction scaffolds is still the difficulty in achieving a perfect match in mechanical properties with normal human auricle cartilage. This problem severely restricts the effectiveness of auricle reconstruction surgery and the improvement of the patient's quality of life. In view of this, the present invention is clinically translation-oriented. According to the measured mechanical property data of normal human auricle cartilage, a medical thermoplastic polyurethane material is selected. Through the thermodynamic property test, pore regulation, and mechanical property test of polyurethane, the processing temperature of polyurethane is finally determined to be 180 °C, and the mass ratio of sodium chloride is 50%. Further, in terms of the material processing method and production, the NaCl porogen method and perfusion molding method are adopted. The former can construct a controllable porous structure, and the latter can reproduce the complex structure of the auricle with high precision. Moreover, both have advantages in terms of cost, performance, etc. Finally, the present invention conducts thermogravimetric analysis, mechanical property test, and creep test on the polyurethane before and after processing, and obtains that the mechanical stability and thermal stability of the polyurethane have not changed.

[0059] Therefore, on the basis of retaining the excellent medical properties of TPU, the present invention optimizes the material by regulating its pore structure, and combines advanced 3D modeling and manufacturing technologies to prepare an auricle cartilage scaffold with mechanical properties adapted to normal human auricle cartilage. Animal experiments have confirmed that the TPU auricle cartilage scaffold prepared by the present invention exhibits excellent biocompatibility and structural stability in the subcutaneous implantation experiment in nude mice. The observation after 1 month of surgery shows that the mechanical properties of the auricle scaffold after implantation and removal are highly matched with normal human auricle cartilage, and the scaffold realizes interface integration with the host tissue, new blood vessel formation, and the inflammatory reaction is controllable.

[0060] Thus, the present invention has for the first time achieved a high degree of adaptation in mechanical properties with normal human auricle cartilage, and thus can provide a safe, efficient and promising solution for clinical auricle reconstruction surgery, and is expected to promote substantial progress in the clinical practice of this field.

[0061] Although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A preparation method of an auricular cartilage scaffold material, characterized in that, Including the following steps: Mix thermoplastic polyurethane and sodium chloride evenly to obtain a mixture; inject the mixture into an auricle mold, perform low-temperature curing treatment, and then demold to obtain a formed material; Wash the formed material to remove sodium chloride to obtain the auricle cartilage scaffold material; in the mixture, the mass percentage of sodium chloride is 25% - 75%.

2. The preparation method of the auricular cartilage scaffold material according to claim 1, wherein, Mix thermoplastic polyurethane and sodium chloride evenly. The specific steps are: heat the thermoplastic polyurethane to melt it to reach a viscous flow state, and then add sodium chloride and stir to mix evenly.

3. The preparation method of the auricular cartilage scaffold material according to claim 2, wherein The temperature of the heating and melting is 170 - 190 °C.

4. The preparation method of the auricular cartilage scaffold material according to claim 3, characterized in that, The temperature of the heating and melting is 180 °C.

5. The preparation method of the auricular cartilage scaffold material according to claim 1, characterized in that, In the mixture, the mass percentage of sodium chloride is 50%.

6. The preparation method of the auricular cartilage scaffold material according to any one of claims 1 to 5, characterized in that, The structure of the auricle mold matches the auricle structure of the application object.

7. The preparation method of the auricular cartilage scaffold material according to any one of claims 1 to 5, characterized in that, The temperature of the low-temperature curing treatment is -10 to -30 °C, and the time of the low-temperature curing treatment is 10 to 30 h.

8. The preparation method of the auricular cartilage scaffold material according to any one of claims 1 to 5, characterized in that, The solvent used for the washing is water, and the washing time is 30 to 100 h; after the washing, drying and equilibration steps are also included.

9. An auricle cartilage scaffold material prepared by the preparation method according to any one of claims 1 - 8.

10. Use of the auricular cartilage scaffold material according to claim 9, characterized in that, Application in the preparation of a human auricle cartilage scaffold.

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