Simulated thrombus and preparation method thereof

Abstract

The invention provides a simulated thrombus and a preparation method thereof. The preparation method comprises the following steps: preparing a polymer matrix solution; preparing gelatin microspheres and composite particles, wherein the composite particles are iron oxide particles coated with polydopamine on the surfaces; mixing the gelatin microspheres and the composite particles with the polymer matrix solution to form a composite dispersion liquid; performing network forming treatment and crosslinking treatment on the composite dispersion liquid to obtain a thrombus precursor; soaking the thrombus precursor in a PBS (Phosphate Buffer Solution) to balance the water content; and performing cyclic compression treatment on the thrombus precursor to obtain the simulated thrombus.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biomedical engineering, and more particularly, to a simulated thrombus and a preparation method thereof. BACKGROUND

[0002] Thrombus is a key factor leading to acute myocardial infarction, ischemic stroke and lower extremity deep vein embolism, and other serious cardiovascular and cerebrovascular diseases. In the prevention and treatment of thrombus, the research and development of thrombus removal devices, vascular biomechanics research and clinical operation simulation training are crucial. However, there are ethical restrictions, scarcity of sources and large individual differences in directly using human or animal thrombus. As a substitute tool, the bionic simulated thrombus can provide an in vitro model of thrombus, but the existing technology has obvious shortcomings in terms of component bionics, mechanical adjustability, preparation repeatability and adhesion characteristics, and the simulation performance is poor, which is difficult to meet the precise needs of the research and development of thrombus removal devices, vascular biomechanics research, and clinical operation simulation training. SUMMARY

[0003] The present application provides a new technical solution for the preparation method of a simulated thrombus, which can at least solve the technical problem of poor simulation performance of the existing simulated thrombus.

[0004] The present application also provides a new technical solution for a simulated thrombus.

[0005] According to a first aspect of the present application, a preparation method of a simulated thrombus is provided, comprising: preparing a polymer matrix solution; preparing gelatin microspheres and composite particles, the composite particles being iron oxide particles coated with polydopamine on the surface; mixing the gelatin microspheres and the composite particles with the polymer matrix solution to form a composite dispersion; performing network forming treatment and crosslinking treatment on the composite dispersion to obtain a thrombus precursor; soaking the thrombus precursor in a PBS buffer to balance the water content; and performing cyclic compression treatment on the thrombus precursor to obtain a simulated thrombus.

[0006] Optionally, in the composite dispersion, the content of the gelatin microspheres and the composite particles is 15% to 18%, and the balance is the polymer matrix solution.

[0007] Optionally, the volume ratio of the gelatin microspheres to the composite particles is 1:1 to 2:1, and the simulated thrombus is an arterial simulated thrombus, and the balanced water content is 40% to 50%.

[0008] Optionally, the volume ratio of the gelatin microspheres to the composite particles is 8:1 to 10:1, and the simulated thrombus is a venous simulated thrombus, and the balanced water content is 60% to 70%.

[0009] Optionally, the polymer matrix solution is a PLGA solution with a concentration of 5% to 10%, a polyvinyl alcohol solution with a concentration of 8% to 12%, or a polyurethane solution with a concentration of 8% to 15%.

[0010] Optionally, the polymer matrix solution is a PLGA solution; and the method for performing network forming treatment and cross-linking treatment on the composite dispersion to obtain the thrombus precursor comprises: pouring the composite dispersion into a mold for freeze-drying to form a porous network structure with a pore size of 50 μm to 200 μm; adding an ultraviolet light-sensitive cross-linking agent accounting for 2% to 5% of the mass of the PLGA to the porous network structure, and performing cross-linking solidification by using ultraviolet light irradiation for 10 min to 20 min to obtain the thrombus precursor.

[0011] Optionally, the polymer matrix solution is a polyvinyl alcohol solution; and the method for performing network forming treatment and cross-linking treatment on the composite dispersion to obtain the thrombus precursor comprises: adding a cross-linking agent to the composite dispersion, and adjusting the pH to 3 to 4 to perform cross-linking reaction to form a pre-cross-linking solution; pouring the pre-cross-linking solution into a mold for freeze-drying to form a porous network structure with a pore size of 50 μm to 200 μm; neutralizing the porous network structure to neutral, and washing with deionized water to remove residues to obtain the thrombus precursor.

[0012] Optionally, the polymer matrix solution is a polyurethane solution; and the method for performing network forming treatment and cross-linking treatment on the composite dispersion to obtain the thrombus precursor comprises: using an electrospinning process to form the composite dispersion into a fiber network structure with a fiber diameter of 500 nm to 1000 nm; and performing cross-linking on the fiber network structure at 60°C to 70°C for 4 h to 6 h to obtain the thrombus precursor; or adding triethylenediamine to the fiber network structure, and performing catalytic cross-linking at 60°C to 70°C for 4 h to 6 h to obtain the thrombus precursor.

[0013] Optionally, the gelatin microspheres have a particle size of 5 μm to 20 μm, and the composite particles have a particle size of 10 nm to 100 nm.

[0014] According to a second aspect of the present application, there is provided a simulated thrombus prepared by any one of the above preparation methods.

[0015] According to the preparation method of the simulated thrombus provided in the application, the polymer matrix simulates the fibrin three-dimensional network framework of the real thrombus, the gelatin microspheres simulate the platelet component in the real thrombus, and the composite particles simulate the red blood cells in the real thrombus, so that the simulated thrombus can be scanned by MRI / CT imaging; the polydopamine on the surface of the composite particles can ensure uniform dispersion of the composite particles in the polymer matrix, and the functional groups on the surface of the polydopamine can be bonded with the polymer matrix, which can significantly enhance the interface bonding strength and structural stability; after the water content is balanced by the PBS buffer, the water content and density of the simulated thrombus can reach the physiological state, which can ensure the simulation performance of the simulated thrombus; after the cyclic compression treatment, the internal stress of the forming can be effectively eliminated, the interface bonding strength between the components can be significantly enhanced, the mechanical properties can be highly matched with the real thrombus, so that the multi-dimensional simulation of the thrombus components, morphology, mechanical properties and imaging ability is realized, and the simulation performance of the simulated thrombus is effectively improved; during the preparation, by adjusting the type and concentration of the polymer matrix, the ratio and cross-linking degree of the gelatin microspheres and the composite particles, the mechanical properties can be widely regulated, so that the mechanical properties of the real thrombus of different pathological types and different anatomical sites can be simulated, and a standardized in-vitro model with highly simulated structure and customizable mechanical properties can be provided for the research and development of thrombus removing instruments, vascular biomechanics research and clinical operation simulation training.

[0016] Other features of the present application, and their advantages, will become apparent in the non-limiting detailed description of exemplary embodiments of the present application, described below with reference to the drawings. BRIEF DESCRIPTION OF DRAWINGS

[0017] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments of the application and, together with the description, serve to explain the principles of the application.

[0018] Figure 1 is a flow chart of a preparation method of a simulated thrombus according to an embodiment provided in the application; Figure 2 is a simulated thrombus prepared in Embodiment 1 of the application; Figure 3 is a simulated thrombus prepared in Embodiment 2 of the application; Figure 4 is a simulated thrombus prepared in Embodiment 3 of the application. DETAILED DESCRIPTION

[0019] Various exemplary embodiments of the present application will now be described in detail with reference to the accompanying drawings. Note that the relative arrangement, numerical expressions, and numerical values of the components and steps set forth in these embodiments are not limiting to the scope of the present application unless otherwise specifically stated.

[0020] The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the scope of this application and its application or use.

[0021] Techniques, methods, and equipment known to those skilled in the art may not be discussed in detail, but where appropriate, such techniques, methods, and equipment should be considered part of the specification.

[0022] In all the examples shown and discussed herein, any specific values ​​should be interpreted as merely exemplary and not as limitations. Therefore, other examples of exemplary embodiments may have different values.

[0023] It should be noted that similar labels and letters in the following figures indicate similar items; therefore, once an item is defined in one figure, it does not need to be discussed further in subsequent figures.

[0024] like Figure 1 As shown, an embodiment of this application provides a method for preparing a simulated thrombus, including steps S100 to S600.

[0025] S100, Prepare polymer matrix solution; S200: Prepare gelatin microspheres and composite particles, wherein the composite particles are iron oxide particles coated with polydopamine. S300: Mix the gelatin microspheres and the composite particles with the polymer matrix solution to form a composite dispersion; S400. The composite dispersion is subjected to network forming and cross-linking treatment to obtain a thrombus precursor. S500. Immerse the thrombus precursor in PBS buffer to balance its water content; S600. The thrombus precursor is subjected to cyclic compression to obtain a simulated thrombus.

[0026] In other words, the simulated thrombus according to the embodiments of this application is mainly prepared by a polymer matrix solution, gelatin microspheres, and composite particles. The polymer matrix solution can be a PLGA solution, a polyurethane solution, or a polyvinyl alcohol solution, and the composite particles are iron oxide particles coated with polydopamine. In preparing the simulated thrombus, the gelatin microspheres and composite particles are first added to the polymer matrix solution and mixed evenly to form a composite dispersion. Subsequently, the composite dispersion undergoes network forming and cross-linking treatments to form a three-dimensional network hydrogel structure, i.e., a thrombus precursor. Next, the thrombus precursor is immersed in PBS buffer to balance its water content. The osmotic pressure of the PBS buffer is consistent with that of blood plasma, which promotes the swelling of the thrombus precursor to a physiologically hydrated state, ensuring a high degree of similarity to a real thrombus and guaranteeing its simulation performance. After the water content of the thrombus precursor was balanced, it was placed on the stage of a tensile testing machine and subjected to 100 cycles of compression at a compression rate of 1 mm / min and a strain of 5%–10% to eliminate internal stress and enhance the interfacial bonding between the gelatin microspheres, composite particles, and polymer matrix, resulting in a highly realistic simulated thrombus. The prepared simulated thrombus has an elastic modulus of 5 kPa–50 kPa, a shear strength of 1 kPa–8 kPa, and a peel adhesion force of 1 N / m–5 N / m, which are highly consistent with the mechanical properties of real thrombi, effectively ensuring its simulation performance.

[0027] Therefore, according to the simulated thrombus preparation method provided in this embodiment, the polymer matrix simulates the fibrin three-dimensional network skeleton of a real thrombus, gelatin microspheres simulate the platelet component in a real thrombus, and composite particles simulate the red blood cells in a real thrombus, enabling the simulated thrombus to be scanned by MRI / CT imaging. The polydopamine on the surface of the composite particles ensures that the composite particles are uniformly dispersed in the polymer matrix, and the functional groups on the surface of the polydopamine can bond with the polymer matrix, which can significantly enhance the interfacial bonding strength and structural stability. After balancing the water content with PBS buffer, the water content and density of the simulated thrombus can reach the physiological state, which can ensure the simulation performance of the simulated thrombus. Then, after cyclic compression... The process effectively eliminates internal stress during molding and significantly enhances the interfacial bonding strength between components, making the mechanical properties highly matched with those of real thrombi. This enables multi-dimensional simulation of thrombus components, morphology, mechanical properties, and imaging capabilities, effectively improving the simulation performance of simulated thrombi. During preparation, by adjusting the type and concentration of the polymer matrix, the ratio of gelatin microspheres to composite particles, and the degree of cross-linking, a wide range of mechanical properties can be controlled. This allows for the simulation of the mechanical properties of real thrombi of different pathological types and anatomical locations, providing standardized in vitro models with customizable mechanical properties and highly realistic structures for the development of thrombus removal devices, vascular biomechanics research, and clinical surgical simulation training.

[0028] In some specific embodiments of this application, the total content of the gelatin microspheres and the composite particles in the composite dispersion is 15wt%~18wt%, with the remainder being the polymer matrix solution.

[0029] In other words, in the composite dispersion, the total content of gelatin microspheres and composite particles is configured to be 15wt%~18wt%, for example, 15wt%, 16wt%, 17wt% and 18wt%, etc., and the content of polymer matrix solution is configured to be 82%~85%, for example, 82wt%, 83wt%, 84wt% and 85wt%, etc.; within this range, the mechanical properties of the simulated thrombus can be highly matched with those of the real thrombus.

[0030] According to one embodiment of this application, the volume ratio of the gelatin microspheres to the composite particles is 1:1 to 2:1, and the simulated thrombus is an arterial simulated thrombus with an equilibrium water content of 40% to 50%.

[0031] Specifically, in preparing simulated arterial thrombi, the volume ratio of gelatin microspheres to composite particles is 1:1 to 2:1, for example, 1:1, 1.2:1, 1.5:1, and 2:1. This ratio range allows the simulated thrombus to achieve an equilibrium water content of 40% to 50% (e.g., 40%, 42%, 45%, and 50%), an elastic modulus of 5 kPa to 50 kPa, a shear strength of 1 kPa to 8 kPa, and an adhesion force of 2 N / m to 5 N / m as determined by the peel test. This is highly consistent with the water content and mechanical properties of real arterial thrombi, thereby effectively ensuring the simulation performance of the simulated thrombus.

[0032] In some specific embodiments of this application, the volume ratio of the gelatin microspheres to the composite particles is 8:1 to 10:1, and the simulated thrombus is a venous simulated thrombus with an equilibrium water content of 60% to 70%.

[0033] In detail, when preparing venous simulated thrombi, the volume ratio of gelatin microspheres to composite particles is 8:1 to 10:1, for example, 8:1, 8.5:1, 9:1, 9.5:1, and 10:1. This ratio range allows the simulated thrombus to achieve an equilibrium water content of 60% to 70% (e.g., 60%, 62%, 64%, 68%, and 70%), an elastic modulus of 5 kPa to 50 kPa, a shear strength of 1 kPa to 8 kPa, and an adhesion force of 1 N / m to 3 N / m as determined by the peel test. This is highly consistent with the water content and mechanical properties of real arterial thrombi, thereby effectively ensuring the simulation performance of the simulated thrombus.

[0034] In some optional examples of this application, the concentration of the polymer matrix solution is 5% to 15%, such as 5%, 8%, 10%, 12% and 15%. Within this range, the mechanical properties and morphological structure of the simulated thrombus can be guaranteed to be highly matched with those of the real thrombus, thereby effectively ensuring the simulation performance of the simulated thrombus.

[0035] According to one embodiment of this application, the polymer matrix solution is a PLGA solution with a concentration of 5% to 10%, a polyvinyl alcohol solution with a concentration of 8% to 12%, or a polyurethane solution with a concentration of 8% to 15%.

[0036] In some examples, the polymer matrix is ​​a PLGA solution. If the concentration of the PLGA solution is too low, the cross-linked network skeleton is too loose, resulting in insufficient mechanical strength and poor structural stability of the simulated thrombus. If the concentration of the PLGA solution is too high, the network is too dense, resulting in excessively low porosity and excessive rigidity, deviating from the viscoelastic characteristics of a real thrombus. In this example, the concentration of the PLGA solution is configured to be 5% w / v to 10% w / v, for example, 5% w / v, 7% w / v, 9% w / v, and 10% w / v. Within this range, the mechanical properties and morphological structure of the simulated thrombus are highly matched with those of a real thrombus, thereby effectively improving the simulation performance.

[0037] In some examples, the polymer matrix is ​​a polyvinyl alcohol (PVA) solution. If the concentration of the PVA solution is too low, the cross-linked network skeleton is too loose, resulting in insufficient mechanical strength and poor structural stability of the simulated thrombus. If the concentration of the PVA solution is too high, the network is too dense, resulting in excessively low porosity and excessive rigidity, deviating from the viscoelastic characteristics of a real thrombus. In this example, the concentration of the PVA solution is configured to be 8% w / v to 12% w / v, for example, 8% w / v, 9% w / v, 10% w / v, and 12% w / v. Within this range, the mechanical properties and morphological structure of the simulated thrombus are highly matched with those of a real thrombus, thereby effectively improving the simulation performance.

[0038] In some examples, the polymer matrix is ​​a polyurethane solution. If the concentration of the polyurethane solution is too low, it indicates that its viscosity is too low. This leads to unstable jetting, excessively fine fiber diameters, and an increase in beaded structures during the subsequent electrospinning process to form the network skeleton, resulting in insufficient mechanical strength and poor structural stability of the fiber network. Conversely, if the concentration of the polyurethane solution is too high, it indicates that its viscosity is too high, making continuous spinning difficult and resulting in excessively large fiber diameters. This leads to excessively low porosity and excessive rigidity, deviating from the viscoelastic characteristics of a real thrombus. In this example, the concentration of the polyurethane solution is configured to be 8% w / v to 15% w / v, for example, 8% w / v, 10% w / v, 10% w / v, and 15% w / v. Within this range, the mechanical properties and morphological structure of the simulated thrombus are highly matched to those of a real thrombus, thereby effectively improving the simulation performance.

[0039] In some specific embodiments of this application, the polymer matrix solution is a PLGA solution; the method for obtaining the thrombus precursor by performing network forming and crosslinking treatment on the composite dispersion is as follows: the composite dispersion is poured into a mold and freeze-dried to form a porous network structure with a pore size of 50μm~200μm; 2%~5% of a UV-sensitive crosslinking agent by mass of PLGA is added to the porous network structure, and crosslinking and curing are performed by irradiation with UV light for 10min~20min to obtain the thrombus precursor.

[0040] In this embodiment, a polyvinyl alcohol solution with a concentration of 5% w / v to 10% w / v is used as the polymer matrix, and a porous network structure with a pore size of 50 μm to 200 μm (e.g., 50 μm, 50 μm, 150 μm, and 200 μm) is constructed using a freeze-drying process. This pore size range closely matches the gaps between blood cell aggregations and the fibrin network channels inside a real thrombus, ensuring that the mechanical properties and morphological structure of the simulated thrombus are highly consistent with those of a real thrombus.

[0041] According to one embodiment of this application, the polymer matrix solution is a polyvinyl alcohol solution; the method for obtaining the thrombus precursor by performing network forming and crosslinking treatment on the composite dispersion is as follows: a crosslinking agent is added to the composite dispersion, and the pH is adjusted to 3-4 to carry out a crosslinking reaction to form a pre-crosslinked solution; the pre-crosslinked solution is poured into a mold and freeze-dried to form a porous network structure with a pore size of 50μm-200μm; the porous network structure is neutralized to neutral and washed with deionized water to remove residues to obtain the thrombus precursor.

[0042] In this embodiment, by using a polyvinyl alcohol solution with a concentration of 8% w / v to 12% w / v as the polymer matrix, and combining it with a controlled crosslinking reaction and freeze-drying process under pH 3 to 4 conditions, a three-dimensional porous network structure with a pore size of 50 μm to 200 μm (e.g., 50 μm, 100 μm, 150 μm, and 200 μm) can be formed. This pore size range is highly consistent with the gaps between blood cell aggregation and fibrin network channels inside a real thrombus, which can ensure that the mechanical properties and morphological structure of the simulated thrombus are highly matched with those of a real thrombus.

[0043] In some specific embodiments of this application, the polymer matrix solution is a polyurethane solution; the method for obtaining the thrombus precursor by performing network forming and crosslinking treatment on the composite dispersion is as follows: the composite dispersion is formed into a fiber network structure with a fiber diameter of 500nm~1000nm using an electrospinning process; the fiber network structure is crosslinked at 60℃~70℃ for 4h~6h to obtain the thrombus precursor; or, triethylenediamine is added to the fiber network structure and catalytically crosslinked at 60℃~70℃ for 4h~6h to obtain the thrombus precursor.

[0044] In this embodiment, by using a polyurethane solution with a concentration of 8% w / v to 15% w / v as the polymer matrix, and combining it with an electrospinning process, a nanofiber network structure with a fiber diameter of 500nm to 1000nm (e.g., 500nm, 600nm, 700nm, 800nm, 900nm, or 1000nm) can be formed, thereby ensuring that the mechanical properties and morphological structure of the simulated thrombus are highly matched with those of the real thrombus.

[0045] According to one embodiment of this application, the gelatin microspheres have a particle size of 5μm to 20μm, and the composite particles have a particle size of 10nm to 100nm.

[0046] In this embodiment, the particle size of the gelatin microspheres is configured to be 5μm~20μm, for example, 5μm, 10μm, 15μm and 20μm; the particle size of the composite particles is configured to be 10nm~100nm, for example, 10nm, 40nm, 60nm, 80nm and 100nm; the combination of this particle size between the gelatin microspheres and the composite separator enables the nanoscale composite particles to effectively fill the gaps between the micron-scale gelatin microspheres, forming a multi-level particle-pore structure, making its mechanical properties, equilibrium water content and microstructure highly consistent with real thrombi, thereby significantly improving the comprehensive simulation performance of simulated thrombi.

[0047] The method for preparing simulated thrombi according to this application will be described in detail below with reference to specific embodiments.

[0048] Example 1 S100. Weigh 5 g of PLGA powder, add dichloromethane / acetone mixed solvent (volume ratio 3:1), stir magnetically for 3 h until completely dissolved, and bring the volume to 100 mL to obtain a 5% w / v PLGA solution. S200. Preparation of gelatin microspheres and composite particles: In preparing gelatin microspheres, 5g of food-grade gelatin powder was added to 100mL of deionized water and stirred in a 60℃ water bath for 20min until dissolved, yielding a 5% w / v gelatin solution. The gelatin solution was then slowly poured into 200mL of soybean oil and magnetically stirred at 800rpm for 12min to form an emulsion. The emulsion was then placed in an ice bath and stirred for another 5min to solidify the microspheres. After filtration and rinsing three times with deionized water, the gelatin microspheres with a particle size of 8μm were obtained. In preparing composite particles, 0.10g of FeCl2 was weighed... 4H2O and 0.27g FeCl3 6H2O was dissolved in 20 mL of deionized water to form a solution. Nitrogen gas was then introduced into the solution for 5 min to remove dissolved oxygen. The solution was then heated to 60 °C and stirred continuously while 1 mol / L NaOH was slowly added dropwise until the pH reached 10.5. The reaction was carried out at a constant temperature for 40 min. After the reaction was completed, the product was centrifuged and washed until the pH was approximately 7 to obtain Fe3O4 particles. The washed product was then redispersed in 50 mL of Tris-HCl buffer solution at pH 8.5. 0.03 g of dopamine hydrochloride was added to the buffer solution and stirred continuously at room temperature for 6 h to form a polydopamine layer on the surface of the Fe3O4 particles. Finally, the particles were centrifuged and dried to obtain composite particles with a particle size of approximately 60 nm. S300, weigh 0.0726g of gelatin microspheres and 0.3774g of composite particles, add them sequentially to 1.35 mL of 5% w / v PLGA solution, then sonicate at 300W for 4 min while maintaining magnetic stirring at 500 rpm, and add 1.2g of deionized water to mix well to obtain the composite solution; S400. The composite solution is poured into a cylindrical silicone mold with a diameter of φ3mm×10mm, pre-frozen at -20℃ for 2h, and then transferred to a vacuum freeze dryer. It is dried at a vacuum of 10Pa and -50℃ for 18h to obtain a porous network structure with a pore size of about 90μm. Subsequently, 1,4-butanediol diacrylate (about 0.00135 g) accounting for 2% of the effective component of PLGA is sprayed into the structure, and cross-linking and curing are carried out by irradiation with 365nm ultraviolet light for 15min to obtain the thrombus precursor. S500. Soak the thrombus precursor in PBS buffer at 37°C and pH 7.4 for 24 hours to allow its water content to reach equilibrium. S600. The thrombus precursor is subjected to 100 cycles of compression at 8% strain and 1 mm / min compression rate on a tensile testing machine to enhance interfacial bonding, thereby obtaining an arterial simulated thrombus prepared with a PLGA matrix. Figure 2 As shown.

[0049] Example 2 S100. Weigh 12g of medical-grade polyurethane, add N,N-dimethylformamide to dissolve it, and then make up to 100mL to obtain a 12% (w / v) polyurethane solution. S200. Preparation of gelatin microspheres and composite particles: In preparing gelatin microspheres, 5g of food-grade gelatin powder was added to 100mL of deionized water and stirred in a 60℃ water bath for 20min until dissolved, yielding a 5% w / v gelatin solution. The gelatin solution was then slowly poured into 200mL of soybean oil and magnetically stirred at 500rpm for 12min to form an emulsion. The emulsion was then placed in an ice bath and stirred for another 5min to solidify the microspheres. After filtration and rinsing three times with deionized water, the microspheres with a particle size of 15μm were obtained. In preparing composite particles, 0.10g of FeCl2 was weighed... 4H2O and 0.27g FeCl3 6H2O was dissolved in 20 mL of deionized water to form a solution. Nitrogen gas was then introduced into the solution for 5 min to remove dissolved oxygen. The solution was then heated to 60 °C and stirred continuously while 1 mol / L NaOH was slowly added dropwise until the pH reached 10.5. The reaction was carried out at a constant temperature for 40 min. After the reaction was completed, the product was centrifuged and washed until the pH was approximately 7 to obtain Fe3O4 particles. The washed product was then redispersed in 50 mL of Tris-HCl buffer solution at pH 8.5. 0.03 g of dopamine hydrochloride was added to the buffer solution and stirred continuously at room temperature for 12 h to form a polydopamine layer on the surface of the Fe3O4 particles. Finally, the particles were centrifuged and dried to obtain composite particles with a particle size of approximately 80 nm. S300: Add 0.932g of gelatin microspheres and 0.508g of composite particles to 1.36mL of 12% w / v polyurethane solution, then sonicate at 300W for 6min, then stir at 500rpm until PDI=0.26, and finally add 5.2g of deionized water and mix well to obtain the composite solution. S400: The composite solution is loaded into a syringe with a 0.8 mm needle, and electrospinning is carried out continuously for 1.5 h under the electrospinning parameters of 18 kV voltage, 15 cm receiving distance, and 0.6 mL / h flow rate, so that the fibers are collected in a cylindrical receiver with a diameter of φ5 mm × 20 mm, and a fiber network structure with a fiber diameter of 800 nm to 900 nm is obtained. Then, the fiber network structure is placed in a 70 °C oven, and 0.1% w / v of triethylenediamine (about 0.0014 mL) based on the volume of polyurethane solution is added. After reacting for 4.5 h, the mixture is cooled and demolded to obtain the thrombus precursor. S500. Soak the thrombus precursor in PBS buffer at 37°C and pH 7.4 for 24 hours to allow its water content to reach equilibrium. S600. The thrombus precursor is subjected to 100 cycles of compression at 8% strain and 1 mm / min compression rate on a tensile testing machine to enhance interfacial bonding, thereby obtaining a venous simulated thrombus prepared with a PLGA matrix. Figure 3 As shown.

[0050] Example 3 S100. Weigh 10g of polyvinyl alcohol, add 100mL of deionized water, heat and stir at 80℃ to dissolve, and obtain a 10% w / v polyvinyl alcohol solution.

[0051] S200. Preparation of gelatin microspheres and composite particles: In preparing gelatin microspheres, 5g of food-grade gelatin powder was added to 100mL of deionized water and stirred in a 60℃ water bath for 20min until dissolved, yielding a 5% w / v gelatin solution. The gelatin solution was then slowly poured into 200mL of soybean oil and magnetically stirred at 1000rpm for 12min to form an emulsion. The emulsion was then placed in an ice bath and stirred for another 5min to solidify the microspheres. After filtration and rinsing three times with deionized water, the microspheres were drained and screened to obtain gelatin microspheres with a particle size of 7μm. In preparing composite particles, 0.10g of FeCl2 was weighed... 4H2O and 0.27g FeCl3 6H2O was dissolved in 20 mL of deionized water to form a solution. Nitrogen gas was then introduced into the solution for 5 min to remove dissolved oxygen. The solution was then heated to 60 °C and stirred continuously while 1 mol / L NaOH was slowly added dropwise until the pH reached 10.5. The reaction was carried out at a constant temperature for 40 min. After the reaction was completed, the product was centrifuged and washed until the pH was approximately 7 to obtain Fe3O4 particles. The washed product was then redispersed in 50 mL of Tris-HCl buffer solution at pH 8.5. 0.03 g of dopamine hydrochloride was added to the buffer solution and stirred continuously at room temperature for 3 h to form a polydopamine layer on the surface of the Fe3O4 particles. Finally, the particles were centrifuged and dried to obtain composite particles with a particle size of approximately 30 nm. S300, weigh 0.25g of gelatin microspheres and 0.55g of composite particles, add them sequentially to 1.86 mL of 10% w / v polyvinyl alcohol solution, then sonicate at 300W for 3 min, then magnetically stir at 500rpm, add 2.25g of deionized water and mix well to obtain the composite solution; S400: Add 1 mL of 1% w / v glutaraldehyde to the composite solution and adjust the pH to 3.5 to carry out the cross-linking reaction, forming a pre-cross-linked solution; inject the pre-cross-linked solution into a cylindrical mold with a φ4mm×15mm diameter, pre-freeze at -20℃ for 2 hours, and then transfer it to a vacuum freeze dryer. Dry it at a vacuum of 10Pa and -50℃ for 20 hours to obtain a porous network structure with a pore size of approximately 50μm~80μm; then immerse the porous network structure in 0.1M NaOH solution to neutralize it to pH=7, and then immerse it in deionized water for 24 hours to remove unreacted glutaraldehyde. Demold the structure to obtain the thrombus precursor. S500. Soak the thrombus precursor in PBS buffer at 37°C and pH 7.4 for 24 hours to allow its water content to reach equilibrium. S600. The thrombus precursor is subjected to 100 cycles of compression at 8% strain and 1 mm / min compression rate on a tensile testing machine to enhance interfacial bonding, thereby obtaining an arterial simulated thrombus prepared with a PLGA matrix. Figure 4 As shown.

[0052] The performance testing methods for Examples 1 to 3 are as follows: 1. Uniaxial compression test The samples were placed in a constant temperature and humidity chamber at 37℃ and 95% humidity for 24 hours to equilibrate (simulating the human physiological environment) to avoid the influence of environmental humidity and temperature on the mechanical properties of the materials. Five parallel samples were prepared for each group, and the average elastic modulus of the five parallel samples was taken as the final result. Then, the samples were compressed to 10% strain at a rate of 0.5 mm / min using a tensile testing machine, and force-displacement data were collected. The elastic modulus (kPa) was calculated by the slope of the linear segment of the stress-strain curve.

[0053] 2. Single shear test A "sandwich" composite sample was used, with a middle layer of simulated thrombus material and upper and lower layers of simulated blood vessel wall material. The simulated thrombus material was tightly bonded to the upper and lower blood vessel wall materials to ensure uniform adhesion (no air bubbles or gaps). The sample was cured at 37℃ for 24 hours to simulate the natural adhesion state of a thrombus to the blood vessel wall. Five parallel samples were prepared for each group, and the average shear strength of the five parallel samples was taken as the final result. The loading rate was set to 1 mm / min, and loading was continuously applied along the shear direction until shear failure occurred. Force-displacement data were collected in real time (sampling frequency 10 Hz). The shear strength (MPa) was calculated using the following formula: τ max = F max / S Where S is the actual area of ​​the shear surface (mm²) 2 ), F max The maximum load is (N).

[0054] 3. Peel test Simulated thrombi were uniformly coated onto the surface of the vessel wall material to a thickness of 3 mm, ensuring no air bubbles and uniform coating. The coating was cured at 37℃ and 95% humidity for 24 hours to allow a stable adhesion interface to form between the thrombus material and the vessel wall material. Five parallel samples were prepared for each group, and the average adhesion strength of the five parallel samples was taken as the final result. Peeling was performed continuously at a rate of 1 mm / min along a 180° direction until the adhesion interface was completely separated. Peeling force-displacement data were collected in real time (sampling frequency 10 Hz). The adhesion strength was calculated using the following formula: τ a = F a e / W Where F a e The average force value during the stable phase of the curve represents the sample adhesion force (N), and W is the sample width (mm), with the unit being N / mm.

[0055] The performance test data for Examples 1 to 3 are shown in Table 1.

[0056] Table 1

[0057] As can be seen from Table 1, the simulated thrombus in Example 1 weighs 3g and has a size of φ3mm×10mm, which is consistent with acute coronary artery thrombosis in humans. It has a dense structure, a water content of 40%, and a mechanical strength of 12kPa. It can be directly used for small-scale in vitro thrombolysis experiments and coronary artery thrombectomy stent compatibility testing.

[0058] Example 2 simulated a thrombus weighing 8g and measuring φ5mm×20mm, matching the chronic deep vein thrombosis of the human lower limb. It has a loose structure, a water content of 65%, and an elastic modulus of 35 kPa, and can be used for the study of the organization process of venous thrombosis and the in vitro evaluation of chronic thrombolytic drugs.

[0059] Example 3 simulates a thrombus weighing 5g and measuring φ4mm×15mm, consistent with chronic carotid artery thrombosis in humans. It has a dense and hard structure, a water content of 45%, and a mechanical strength of 42 kPa. It can be used for performance testing of carotid artery interventional devices and simulated thrombotic imaging (MRI / CT) scans.

[0060] In summary, the simulated thrombus preparation method provided in this embodiment uses a polymer matrix to simulate the three-dimensional fibrin network skeleton of a real thrombus, gelatin microspheres to simulate platelet components in a real thrombus, and composite particles to simulate red blood cells in a real thrombus, enabling the simulated thrombus to be scanned by MRI / CT imaging. The polydopamine on the surface of the composite particles ensures uniform dispersion of the particles in the polymer matrix, and the functional groups on the polydopamine surface can bond with the polymer matrix, significantly enhancing interfacial bonding strength and structural stability. After equilibration with PBS buffer, the water content and density of the simulated thrombus reach physiological levels, ensuring the simulation performance of the thrombus. Further cyclic pressure... Shrinkage treatment can effectively eliminate internal stress during molding and significantly enhance the interfacial bonding strength between components, making the mechanical properties highly matched with those of real thrombi. This enables multi-dimensional simulation of thrombus components, morphology, mechanical properties, and imaging capabilities, effectively improving the simulation performance of simulated thrombi. During preparation, by adjusting the type and concentration of the polymer matrix, the ratio of gelatin microspheres to composite particles, and the degree of cross-linking, a wide range of mechanical properties can be controlled. This allows for the simulation of the mechanical properties of real thrombi of different pathological types and anatomical locations, providing standardized in vitro models with customizable mechanical properties and highly realistic structures for the development of thrombus removal devices, vascular biomechanics research, and clinical surgical simulation training.

[0061] This application also provides a simulated thrombus, which is prepared by the preparation method described in any of the above embodiments. Since the preparation method of the simulated thrombus according to the embodiments of this application has the above-mentioned technical effects, the simulated thrombus according to the embodiments of this application also has corresponding technical effects, which will not be repeated in this embodiment.

[0062] While specific embodiments of this application have been described in detail by way of examples, those skilled in the art should understand that the above examples are for illustrative purposes only and are not intended to limit the scope of this application. Those skilled in the art should understand that modifications can be made to the above embodiments without departing from the scope and spirit of this application. The scope of this application is defined by the appended claims.

Claims

1. A method for preparing a simulated thrombus, characterized in that, include: Preparation of polymer matrix solution; Gelatin microspheres and composite particles were prepared, wherein the composite particles were iron oxide particles coated with polydopamine. The gelatin microspheres and the composite particles are mixed with the polymer matrix solution to form a composite dispersion. The composite dispersion was subjected to network forming and cross-linking treatment to obtain a thrombus precursor; The thrombus precursor was immersed in PBS buffer to equilibrate its water content; The thrombus precursor was subjected to cyclic compression to obtain a simulated thrombus.

2. The preparation method according to claim 1, characterized in that, In the composite dispersion, the total content of the gelatin microspheres and the composite particles is 15wt%~18wt%, with the remainder being the polymer matrix solution.

3. The preparation method according to claim 2, characterized in that, The volume ratio of the gelatin microspheres to the composite particles is 1:1 to 2:1, and the simulated thrombus is an arterial simulated thrombus with an equilibrium water content of 40% to 50%.

4. The preparation method according to claim 2, characterized in that, The volume ratio of the gelatin microspheres to the composite particles is 8:1 to 10:1, and the simulated thrombus is a venous simulated thrombus with an equilibrium water content of 60% to 70%.

5. The preparation method according to claim 1, characterized in that, The polymer matrix solution is a PLGA solution with a concentration of 5% to 10%, a polyvinyl alcohol solution with a concentration of 8% to 12%, or a polyurethane solution with a concentration of 8% to 15%.

6. The preparation method according to claim 5, characterized in that, The polymer matrix solution is a PLGA solution; the method for obtaining the thrombus precursor by performing network forming and crosslinking treatment on the composite dispersion is as follows: The composite dispersion was poured into a mold and freeze-dried to form a porous network structure with a pore size of 50μm~200μm. Add 2% to 5% of the mass of PLGA to the porous network structure and irradiate with ultraviolet light for 10 to 20 minutes to crosslink and cure, thereby obtaining the thrombus precursor.

7. The preparation method according to claim 5, characterized in that, The polymer matrix solution is a polyvinyl alcohol solution; the method for obtaining the thrombus precursor by performing network forming and crosslinking treatment on the composite dispersion is as follows: A crosslinking agent is added to the composite dispersion, and the pH is adjusted to 3-4 to carry out a crosslinking reaction, forming a pre-crosslinked solution; The pre-crosslinked solution was poured into a mold and freeze-dried to form a porous network structure with a pore size of 50 μm to 200 μm. The porous network structure is neutralized to neutral and washed with deionized water to remove residues, thus obtaining the thrombus precursor.

8. The preparation method according to claim 5, characterized in that, The polymer matrix solution is a polyurethane solution; the method for obtaining the thrombus precursor by performing network forming and crosslinking treatment on the composite dispersion is as follows: The composite dispersion was formed into a fiber network structure with a fiber diameter of 500 nm to 1000 nm using an electrospinning process. The fiber network structure is cross-linked by maintaining it at 60℃~70℃ for 4h~6h to obtain a thrombus precursor; or, Triethylenediamine was added to the fiber network structure, and catalytic cross-linking was carried out at 60℃~70℃ for 4h~6h to obtain the thrombus precursor.

9. The preparation method according to any one of claims 1-8, characterized in that, The gelatin microspheres have a particle size of 5μm to 20μm, and the composite particles have a particle size of 10nm to 100nm.

10. A simulated thrombus, characterized in that, The simulated thrombus is prepared by the preparation method according to any one of claims 1-9.

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