Method for preparing endothelialized vascular prosthesis

Abstract

The application provides a preparation method of an endothelialized artificial blood vessel, which comprises the following steps: firstly, preparing the artificial blood vessel by using polyvinyl alcohol together with a blood vessel base material; secondly, removing the polyvinyl alcohol from the artificial blood vessel by using the solubility of the polyvinyl alcohol, so as to form a hole structure in the blood vessel; and thirdly, processing by using an extrusion tapping process, so as to form a thread convex on the inner wall of the blood vessel, and the thread convex and the hole structure form a topological structure together, endothelial cells are captured by using the topological structure, the impact of fluid on the cells is reduced, the cells can better adhere to the inner wall of the blood vessel, compared with a traditional method of attracting endothelial cells by using grafting or chemical modification, the cells are more beneficial to adhere to the inner wall of the blood vessel, and the endothelialization speed is accelerated.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological materials, and particularly relates to a preparation method of endothelialized artificial blood vessels. BACKGROUND

[0002] Endothelialization of an artificial blood vessel refers to forming a membrane-like tissue layer of endothelial cells on the inner wall of the artificial blood vessel. The formation of the layer can avoid adhesion of platelets and leukocytes on the inner wall of the blood vessel, is beneficial to maintaining the normal function of the blood vessel, makes the function and structure of the artificial blood vessel close to the natural blood vessel, and is beneficial to promoting healing and recovery of the blood vessel.

[0003] In the prior art, a patent document with the application number CN202210247352.1 and the publication date of September 2, 2022 and the name of “artificial blood vessel with double functions of promoting endothelialization and anticoagulation and preparation method, use” loads RGD-pH responsive silica drug-loaded nanoparticles on the outer wall of the inner layer of the artificial blood vessel, which can realize slow and stable release of the drug under normal pH of the human body blood, and the RGD polypeptide can induce ordered growth of endothelial cells, and the silica nanoparticles can also be degraded in vivo, which is beneficial to autologous blood vessel regeneration; the loaded anticoagulant drug can long-acting prevent thrombosis in the artificial blood vessel and improve the blood compatibility of the artificial blood vessel. However, due to the rapid flow of cells in the blood, there is a shear force in the flowing liquid, the greater the flow rate, the greater the shear force, and the endothelial cells cannot be fixed, which is not conducive to the adhesion, growth and proliferation of the endothelial cells.

[0004] In view of this, it is necessary to design an improved preparation method of endothelialized artificial blood vessels to solve the above problems. SUMMARY

[0005] The present application relates to the technical field of biological materials, and particularly relates to a preparation method of endothelialized artificial blood vessels.

[0006] To achieve the above-mentioned application purposes, the present application provides a preparation method of endothelialized artificial blood vessels, comprising the following steps:

[0007] S1, dissolving polyvinyl alcohol and a blood vessel substrate in an organic solvent to form a uniform and stable mixed solution; then coating the mixed solution on a blood vessel mold to obtain a tubular artificial blood vessel; and then dissolving the polyvinyl alcohol in the artificial blood vessel to form a pore structure on the artificial blood vessel, and the porosity of the artificial blood vessel is 60-80%;

[0008] S2, the inner wall of the artificial blood vessel with the hole structure obtained in step S1 is treated by using an extrusion tapping process to form thread protrusions on the inner wall of the artificial blood vessel with the hole structure, and the thread protrusions and the hole structure on the thread protrusions together constitute a topological structure; then, the cell suspension obtained after the subculture digestion is injected into the artificial blood vessel with the topological structure, and in the process of flowing, the cells in the cell suspension enter and stay in the hole structure of the inner wall of the artificial blood vessel with the topological structure, and finally grow into an endothelial cell layer; and after decellularization treatment, cleaning and sterilization treatment, an endothelialized artificial blood vessel with a smooth inner wall is prepared, and the porosity of the endothelialized artificial blood vessel is 20-50%.

[0009] Preferably, in step S1, the mass ratio of the vascular substrate to the polyvinyl alcohol in the mixed solution is (95:5)-(80:20).

[0010] Preferably, in step S1, the vascular substrate is polyurethane or polycaprolactone.

[0011] Preferably, in step S1, the organic solvent is N,N-dimethylformamide or dimethyl sulfoxide.

[0012] Preferably, in step S2, the height of the thread protrusion is 2-6mm, the thickness is 0.5-2mm, the pitch is 0.5-1mm, and the tooth type angle is 60-120°.

[0013] Preferably, in step S2, the hole diameter of the hole structure in the thread protrusion is 20-50μm.

[0014] Preferably, in step S1, the inner diameter of the artificial blood vessel is 6-30mm, and the wall thickness is 0.3-1mm.

[0015] Preferably, in step S1, the polyvinyl alcohol is dissolved by immersing the artificial blood vessel in water at a temperature of 95-100℃ for 5-8h, and the mass ratio of water to the artificial blood vessel during the immersion is greater than 1000.

[0016] The beneficial effects of the present application are:

[0017] 1. The preparation method of the endothelialized artificial blood vessel provided by the application, which comprises the following steps: preparing an artificial blood vessel by using polyvinyl alcohol together with a blood vessel base material, removing the polyvinyl alcohol from the artificial blood vessel by utilizing the solubility of the polyvinyl alcohol to form a hole structure in the blood vessel, and processing by using an extrusion tapping process to form a thread protrusion on the inner wall of the blood vessel, wherein the thread protrusion and the hole structure together form a topological structure, the formation of the topological structure can effectively increase the specific surface area of the inner wall of the blood vessel, and is beneficial to providing an attachment point for cells, attaching more cells and accelerating the endothelialization speed in the process of endothelialization; meanwhile, the porosity of the blood vessel is regulated by controlling the mixing ratio of the polyvinyl alcohol and the blood vessel base material in the artificial blood vessel, so that a proper amount of cells are intercepted in the hole structure in the process of endothelialization, and the thickness of the final obtained membrane-shaped tissue layer is uniform. Through the above-mentioned manner, the endothelialization time is greatly shortened, the endothelialization work efficiency is effectively improved, and the endothelialized artificial blood vessel with a uniform and smooth inner wall is prepared.

[0018] 2. The preparation method of the endothelialized artificial blood vessel provided by the application, which comprises the following steps: forming a topological structure formed by the thread protrusion and the hole structure on the inner wall of the artificial blood vessel, and capturing endothelial cells by utilizing the topological structure, so as to reduce the impact of fluid on the cells and enable the cells to better adhere to the inner wall of the blood vessel, compared with the traditional method of attracting endothelial cells by using grafting or chemical modification, the cells are more beneficial to adhere to the inner wall of the blood vessel; secondly, the hole structure distributed on the inner wall of the blood vessel can form a planar structure in the axial direction of the blood vessel, and after the endothelial cells grow on the planar structure, a continuous membrane layer can be formed, so as to accelerate the endothelialization speed, meanwhile, the layered structure of the membrane-shaped tissue layer in the process of endothelialization can be avoided, and the stability and biocompatibility of the blood vessel are enhanced; the artificial blood vessel is subjected to the endothelialization treatment by utilizing the endothelial cells to form the membrane-shaped tissue layer, the artificial blood vessel is subjected to the endothelialization in a bionic manner, and finally the performance and structure of the prepared artificial blood vessel are closer to the human body blood vessel, and the practicality of the artificial blood vessel in actual application is improved. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 The structure schematic diagram of the endothelialized artificial blood vessel prepared by the preparation method of the endothelialized artificial blood vessel provided by the application;

[0020] Figure 2 The structure schematic diagram of the endothelialized artificial blood vessel prepared by the preparation method of the endothelialized artificial blood vessel provided by the application; Figure 1 The structure schematic diagram of the endothelialized artificial blood vessel prepared by the preparation method of the endothelialized artificial blood vessel provided by the application; DETAILED DESCRIPTION

[0021] In order to make the purpose, technical scheme and advantages of the application more clear, the application is described in detail below with reference to the drawings and specific embodiments.

[0022] It should be noted that, in order not to obscure the present application with unnecessary details, only the structures and / or processing steps closely related to the present application are shown in the drawings, and other details not closely related to the present application are omitted.

[0023] In addition, it should be noted that the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements not only includes those elements, but also includes other elements not explicitly listed or inherent to such a process, method, article or device.

[0024] Please refer to Figures 1 to 2 As shown in the drawings, the present application provides a preparation method of endothelialized artificial blood vessels, comprising the following steps:

[0025] S1, dissolving polyvinyl alcohol and a vascular substrate in an organic solvent to form a uniform and stable mixed solution; then coating the mixed solution on a blood vessel mold to obtain a tubular artificial blood vessel; then immersing the artificial blood vessel in water at a temperature of 95-100°C for 5-8h to dissolve the polyvinyl alcohol in the artificial blood vessel, i.e. to form a pore structure on the artificial blood vessel, and the porosity of the pore structure on the artificial blood vessel is 60-80%;

[0026] S2, using an extrusion tapping process to process the inner wall of the artificial blood vessel with the pore structure obtained in step S1 to form a thread protrusion on the inner wall of the blood vessel, since the thread protrusion is formed by extruding the inner wall of the blood vessel, the original pore structure is still maintained on the thread protrusion, and the thread protrusion and the pore structure of the inner wall together constitute a topological structure, which effectively increases the specific surface area of the inner wall of the blood vessel, the endothelialization process can provide more attachment points for cells, and the speed of endothelialization is accelerated; then, injecting the cell suspension obtained after subculture and digestion into the inside of the artificial blood vessel with the topological structure, in the process of flowing, the cells in the cell suspension enter and stay in the pore structure of the inner wall of the blood vessel, and finally grow into an endothelial cell layer, and then after decellularization treatment, washing and sterilization treatment, an endothelialized artificial blood vessel with a smooth inner wall and a porosity of 20-50% is prepared.

[0027] Preferably, in step S1, the vascular substrate is polyurethane, polycaprolactone, etc.; the content ratio of the vascular substrate and polyvinyl alcohol in the mixed solution is (95:5)-(80:20), and after mixing the vascular substrate and polyvinyl alcohol according to the above ratio, it can be ensured that the porosity of the pore structure in the subsequently prepared artificial blood vessel is within a suitable range.

[0028] Preferably, in step S1, the organic solvent is N,N-dimethylformamide or dimethyl sulfoxide.

[0029] Preferably, in step S1, the inner diameter of the artificial blood vessel is 6-30 mm, and the wall thickness is 0.3-1 mm.

[0030] Preferably, in step S2, the height of the thread convex is 2-6 mm, the thickness is 0.5-2 mm, the pitch is 0.5-1 mm, and the tooth angle is 60-120°.

[0031] Preferably, in step S2, the hole diameter of the hole structure in the thread convex is 20-50 μm, which is beneficial to the entry of endothelial cells and avoids the loss of endothelial cells due to inappropriate hole size, thereby affecting the endothelialization process.

[0032] Preferably, in step S2, the distribution density of the thread convex in the artificial blood vessel is 5-20 pieces / cm, and the porosity of the artificial blood vessel is 20-50%. The porosity is related to the amount of polyvinyl alcohol used in the preparation process. When the porosity is less than 20%, the specific surface area of the inner wall of the blood vessel is limited, and the purpose of accelerating the endothelialization process cannot be achieved. When the porosity is higher than 50%, the number of cells deposited during the endothelialization process is too large, and even cell accumulation occurs, resulting in uneven membrane tissue layer and affecting the endothelialization effect.

[0033] The preparation method of the endothelialized artificial blood vessel is further described below in combination with specific examples.

[0034] Example 1

[0035] An endothelialized artificial blood vessel is prepared by the following steps:

[0036] S1, polyvinyl alcohol and polyurethane are dissolved in N, N-dimethylformamide to form a uniform and stable mixed solution, and the solution concentration is 15%. The mass ratio of polyvinyl alcohol to polyurethane in the mixed solution is 15:85. Then the above mixture is coated on a blood vessel mold to obtain a tubular artificial blood vessel, and the inner diameter of the artificial blood vessel is 8 mm and the wall thickness is 0.5 mm. Then the artificial blood vessel is placed in water at a temperature of 98°C, so that the polyvinyl alcohol in the artificial blood vessel is dissolved, i.e. a hole structure is formed on the artificial blood vessel, the average hole diameter of the hole structure is 35 μm, and the porosity of the artificial blood vessel is 70%.

[0037] S2, the inner wall of the artificial blood vessel with the hole structure obtained in step S1 is treated by using an extrusion tapping process to form thread protrusions on the inner wall of the blood vessel, the height of the thread protrusions is 0.6 mm, the thickness is 0.7 mm, and the pitch is 1 mm, and the thread protrusions and the hole structure of the inner wall together form a topological structure; then, the cell suspension obtained after the subculture digestion is injected into the artificial blood vessel with the topological structure, the cells in the cell suspension enter and stay in the hole structure of the inner wall of the blood vessel in the process of flowing, and finally grow into an endothelial cell layer, and after decellularization treatment, cleaning and sterilization treatment, an endothelial artificial blood vessel is prepared, the porosity of the hole structure in the blood vessel is 35%, and the endothelial time is 10 days, and a schematic diagram of the artificial blood vessel prepared in the embodiment is shown in FIGS. Figure 1 and Figure 2 As can be seen from the figure, the inner wall of the artificial blood vessel prepared by using the method of the embodiment is smooth.

[0038] Comparative Example 1

[0039] The difference between Comparative Example 1 and Example 1 is only that the inner wall of the artificial blood vessel with the hole structure is not treated by using the extrusion tapping process, but the cell suspension is directly injected to perform endothelial treatment on the blood vessel, and the other steps are basically the same as those of Example 1, which will not be described here. The results show that the endothelial treatment time by using the above-mentioned method is more than 30 days, which is obviously longer than 10 days in Example 1, because the artificial blood vessel formed by using the method of Example 1 has a topological structure formed by the thread protrusions and the hole structure, and the formation of the thread protrusions is beneficial to the interception of the cells in the cell suspension and the entry of the cells into the hole structure, which accelerates the speed of cell adhesion on the inner wall of the blood vessel and greatly shortens the endothelial time.

[0040] In summary, the preparation method of the endothelial artificial blood vessel provided by the present application first uses polyvinyl alcohol to prepare an artificial blood vessel together with a blood vessel substrate, then uses the solubility of polyvinyl alcohol to remove it from the artificial blood vessel to form a hole structure in the blood vessel; then, an extrusion tapping process is used to treat the inner wall of the blood vessel to form thread protrusions, and the thread protrusions and the hole structure together form a topological structure, the formation of the topological structure can effectively increase the specific surface area of the inner wall of the blood vessel, which is beneficial to providing attachment points for cells in the process of endothelialization, attaching more cells, accelerating the speed of endothelialization, and forming a smooth and uniform membrane-like tissue layer on the inner wall of the artificial blood vessel.

[0041] The above embodiments are only used to illustrate the technical solutions of the present application and not to limit it, although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present application can be modified or replaced equivalently without departing from the spirit and scope of the technical solutions of the present application.

Claims

1. A method for preparing an endothelialized artificial blood vessel, characterized in that: The steps include: S1. Dissolving polyvinyl alcohol and a vascular base material in an organic solvent to form a uniform and stable mixed solution; applying the mixed solution to a vascular mold to obtain a tubular artificial blood vessel; dissolving the polyvinyl alcohol in the artificial blood vessel to form a porous structure on the artificial blood vessel, wherein the porosity of the artificial blood vessel is 60-80%; the mass ratio of the vascular base material to the polyvinyl alcohol in the mixed solution is (95:5)-(80:20); S2. Processing the inner wall of the artificial blood vessel with the hole structure obtained in step S1 by an extrusion tapping process to form a threaded protrusion on the inner wall of the artificial blood vessel with the hole structure, wherein the threaded protrusion and the hole structure on the threaded protrusion together constitute a topological structure; Then, the cell suspension obtained after passage and digestion is injected into the artificial blood vessel with a topological structure. During the flow of the cell suspension, the cells in the cell suspension enter and remain in the pore structure of the inner wall of the artificial blood vessel with the topological structure, and eventually grow into an endothelial cell layer. After decellularization, cleaning and sterilization, an endothelialized artificial blood vessel with a smooth inner wall is obtained. The porosity of the endothelialized artificial blood vessel is 20-50%. The height of the thread protrusion is 2-6 mm, the thickness is 0.5-2 mm, the pitch is 0.5-1 mm, and the tooth angle is 60-120°.

2. The method for preparing an endothelialized artificial blood vessel according to claim 1, wherein: In step S1, the vascular substrate is polyurethane or polycaprolactone.

3. The method for preparing an endothelialized artificial blood vessel according to claim 1, wherein: In step S1, the organic solvent is N,N-dimethylformamide or dimethyl sulfoxide.

4. The method for preparing an endothelialized artificial blood vessel according to claim 1, wherein: In step S2, the hole structure in the threaded protrusion has a pore size of 20-50 μm.

5. The method for preparing an endothelialized artificial blood vessel according to claim 1, wherein: In step S1, the inner diameter of the artificial blood vessel is 6-30 mm, and the wall thickness is 0.3-1 mm.

6. The method for preparing an endothelialized artificial blood vessel according to claim 1, wherein: In step S1, the polyvinyl alcohol is dissolved in the following manner: the artificial blood vessel is immersed in water at a temperature of 95-100°C for 5-8 hours, wherein the mass ratio of water to the artificial blood vessel is greater than 1000 during the immersion.

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