Preparation method of a collagen-based bionic small-caliber artificial blood vessel and the artificial blood vessel

Abstract

The present invention belongs to the technical field of artificial blood vessels, and specifically relates to a preparation method of a collagen-based bionic small-diameter artificial blood vessel and an artificial blood vessel. A high-concentration collagen solution and a low-concentration collagen solution are respectively prepared and crosslinked. In three sub-cavities of a die cavity, the high-concentration collagen solution is injected into the two side cavities, and the low-concentration collagen solution is injected into the middle cavity. After the collagen systems on both sides are fully air-dried into films, the collagen system in the middle is freeze-dried, and then wound on a base tube. During the winding process, a high-concentration collagen solution is applied between each winding layer for adhesion, so as to form an artificial blood vessel with a tube wall structure sequentially comprising a first collagen-based film layer, a collagen-based sponge layer, and a second collagen-based film layer from the inside to the outside.

Description

Technical Field

[0001] The present invention belongs to the technical field of artificial blood vessels, and particularly relates to a preparation method of a collagen-based bionic small-caliber artificial blood vessel and an artificial blood vessel. Background Art

[0002] Cardiovascular diseases have become a major public health problem. The number of people who die from cardiovascular diseases globally each year ranks first among various causes of death, seriously threatening human health. When the blood vessels of the human body itself are severely stenosed or occluded, vascular transplantation is the most effective method. However, due to the limited number of autologous blood vessels and the immune rejection reaction of allogeneic blood vessels, the research and development of medical artificial blood vessels have very important practical significance.

[0003] Artificial blood vessels are substitutes for many severely stenosed or occluded blood vessels, and are mostly vascular substitutes synthesized from high-molecular materials such as nylon, polyester (Dacron), and polytetrafluoroethylene (PTFE). They are suitable for vascular bypass surgeries throughout the body. Large and medium-caliber artificial blood vessels (>6 mm) have been widely used in tissue repair and vascular reconstruction surgeries, and good therapeutic effects have been achieved clinically. In contrast, small-caliber blood vessels (≤6 mm) are in a special state of high tension and low blood flow. Clinically, they mainly show poor biocompatibility, and their compliance cannot match that of autologous blood vessels, which easily causes intimal hyperplasia and thrombosis, etc., restricting their clinical application. However, peripheral diseased artery replacement, coronary artery bypass grafting of the heart, etc. all require the use of small-caliber artificial blood vessels. It can be seen that there is a huge demand for small-caliber artificial blood vessels clinically. Therefore, the construction of small-caliber artificial blood vessels needs to be broken through.

[0004] As an important component of the extracellular matrix, collagen is the most widely distributed structural protein in animals and is also an important object for the comprehensive utilization of biological resources. It has low immunogenicity and contains biological information such as cell adhesion peptides (RGD) and specific cell adhesion signals, playing an important role in cell migration and tissue development. Secondly, in the blood vessel wall, collagen is a reticular structure formed by the interweaving of connective tissue, elastin, and polysaccharide proteins, which can accelerate the metabolism of the blood vessel wall, maintain the elasticity of the blood vessel wall, ensure normal blood flow, and prevent blood vessel rupture and embolism. Collagen also has a coagulation effect. After collagen acts on platelets, a series of subsequent processes associated with blood aggregation are triggered, thereby rapidly coagulating the blood. In view of this, collagen-based materials have broad application prospects in the field of vascular tissue engineering.

[0005] However, the aggregated structure of natural collagen is destroyed during the dissolution process, resulting in the loss of the originally excellent properties of collagen. At the same time, the natural blood vessel wall is mainly composed of the intima, media, and adventitia, and each layer of the membrane is different. And the texture of collagen after forming is single, so there is still room for further improvement. Summary of the Invention

[0006] To solve the above technical problems, the present invention provides a method for preparing a collagen-based bionic small-caliber artificial blood vessel:

[0007] (1) Prepare a high-concentration collagen solution and a low-concentration collagen solution respectively, and crosslink each collagen solution through 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS);

[0008] (2) Install a partition to divide the mold cavity with an upward opening into three cavities arranged in sequence. Inject the high-concentration collagen solution after crosslinking treatment in step (1) into the two side cavities respectively, and inject the low-concentration collagen solution after crosslinking treatment in step (1) into the middle cavity. When the collagen solutions in each cavity are naturally air-dried to a non-flowing gel state, remove the partition from the mold cavity. After the collagen gel systems on both sides are further air-dried into films, perform freeze-drying on the collagen gel system in the middle;

[0009] (3) After demolding the two-dimensional collagen film obtained in step (2), parallel to the base tube along the edge away from its freeze-dried area, and wind it on the base tube to obtain a three-dimensional multi-layer tube. During the winding process, apply a high-concentration collagen solution between layers to bond the layers together;

[0010] (4) After air-drying the three-dimensional multi-layer tube obtained in step (3), remove the base tube to leave the artificial blood vessel.

[0011] Preferably: In step (1), the mass concentration of the high-concentration collagen solution is 7.5% - 9.5%, and the mass concentration of the low-concentration collagen solution is 5.5% - 7.0%.

[0012] Preferably: In step (1), first disperse 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) in deionized water at a concentration of 15 mg / mL respectively, and then add the dispersion of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and the dispersion of N-hydroxysuccinimide (NHS) to each collagen solution, and mix and stir at 4°C for 24 h to achieve crosslinking treatment.

[0013] Furthermore, the mass ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) to collagen is 1:5.0 - 6.5, and the mass ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) to N-hydroxysuccinimide (NHS) is 1 - 1.2:0.8 - 1.2.

[0014] Preferably: In step (2), the mold cavity is a cuboid cavity with an upward opening, 6 cm in length, 4 - 5 cm in width. Two partitions are vertically inserted and arranged in the mold cavity to divide the mold cavity into three cavities along the width direction. The thickness of the partition is 0.23 mm.

[0015] The liquid level height of the low-concentration collagen solution injected into the central cavity is 1.2 to 2.5 times that of the high-concentration collagen solution injected into any one of the two side cavities. Among them, the liquid level heights of the high-concentration collagen solutions injected into the two side cavities are the same;

[0016] Furthermore, when the collagen gel systems on both sides are fully air-dried to form films, 80% to 90% of the water in the central collagen gel system has also evaporated, and it can continue to maintain a non-flowing gel state.

[0017] Preferably: In step (3), the base tube is a rigid plastic catheter with an outer tube diameter (diameter) of 2 to 5 mm.

[0018] The present invention also provides an artificial blood vessel prepared by the above method.

[0019] Preferably: The tube wall of the artificial blood vessel includes a three-layer structure, which are, from the inside to the outside, a first collagen-based thin film layer, a collagen-based sponge layer, and a second collagen-based thin film layer;

[0020] Furthermore, the thickness of the collagen-based sponge layer is 1 to 2 mm.

[0021] The beneficial effects of the present application are as follows:

[0022] Aiming at the problem that the aggregated structure of natural collagen is damaged during the dissolution process, resulting in the loss of the original excellent properties of collagen, EDC-NHS is added as a cross-linking agent in the present application, so that amide bonds are formed between the amino groups and carboxyl groups on the collagen molecules, thereby improving the mechanical properties and structural stability of the collagen-based artificial blood vessel.

[0023] The natural blood vessel wall consists of the intima, media, and adventitia. Among them, the intima is the thinnest layer. The media is a loose tissue mainly composed of elastic membranes and smooth muscles. There are intermediate junctions and gap junctions between smooth muscle fibers, which can serve as the only type of channel for communicating adjacent cell plasma membranes and provide a channel for the direct exchange of intercellular signaling molecules such as particles, small molecule metabolites, and second messengers. Therefore, in this solution, a dense and uniform collagen-based thin film is creatively used as the inner and outer layers of the artificial blood vessel wall, and a collagen-based sponge with a loose pore structure is used as the middle membrane layer of the artificial blood vessel wall, thus forming a three-layer structure bionic blood vessel with a loose middle and dense inner and outer sides. Through the porous lumen structure of the collagen sponge, the infiltration and penetration of cells and tissues around the blood vessel graft are enhanced, and at the same time, an anchoring effect is achieved, providing more space and nutrient transportation for cell growth, thereby improving the tissue compatibility of the collagen-based artificial blood vessel. After transplantation, it has good metabolic functions, is not easy to form thrombus, has a satisfactory long-term patency rate, and helps the growth and proliferation of tissue cells such as endothelial cells and smooth muscle cells on the artificial blood vessel (meanwhile, the implanted artificial blood vessel gradually degrades in the body and is gradually replaced by newly grown tissue), showing the activity of inducing tissue repair cell movement and migration into the scaffold;

[0024] The prepared artificial blood vessel has a certain strength and anti-bending flexibility, is suitable for replacing blood vessels at curved parts in the body, and has good suture performance during bypass surgery.

[0025] In the preparation method, the mold cavity is separated by a manually inserted partition board in this application. Therefore, it is impossible to achieve complete sealing between the bottom edge of the partition board and the bottom of the mold cavity groove. During the air-drying and curing process of the collagen solution in each cavity, a small range of mutual penetration will occur through this channel, and then they will adhere and combine together to form an integral two-dimensional collagen thin film (which is convenient for subsequent winding into a tube operation on the base tube), and at the same time, it does not affect the distribution of different collagen matrices in the upper layer;

[0026] At the same time, when it is naturally air-dried until the collagen solution in each cavity is in a non-flowing gel state, the partition board is immediately removed from the mold cavity, rather than waiting until the collagen in both cavities is completely air-dried before removal. This is because at this time, under the condition of unchanged macroscopic shape, a small degree of mutual adhesion and combination will still occur between the collagen gels in adjacent cavities, which is beneficial to improving the connection strength between the collagen in adjacent cavities. If the partition board is removed after the collagen on both sides is completely air-dried, this combined effect will be greatly reduced;

[0027] Meanwhile, the concentrations of the collagen solutions poured into the respective cavities are controlled to be different: the high-concentration collagen solution contains relatively less water. Therefore, when the water in it has completely volatilized and is fully air-dried and solidified, the low-concentration collagen solution still contains a certain amount of solvent water. At this time, instead of the previous natural air-drying operation, the collagen dispersion system still containing water in the middle cavity is freeze-dried. Compared with natural air-drying, freeze-drying directly vaporizes and removes the frozen solvent water in the system. Therefore, rich pore structures are left on the collagen gel system, and the thickness change of the system is very small, finally forming a sponge structure similar to a porous one, thus having the aforementioned "collagen-based sponge layer".

[0028] On this basis, the liquid level height of the solution in the middle cavity is greater than that of the two side cavities. On the one hand, this is also to facilitate film formation first on both sides during the air-drying process because the liquid level heights of the solutions on both sides are low. Moreover, the liquid level height of the middle solution is high, so that it will not be air-dried too quickly resulting in too thin a thickness in the middle area. After a collagen gel with an appropriate thickness is formed in the middle area, it is then freeze-dried into a collagen-based sponge scaffold. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Schematic diagram of the layer structure of the collagen-based bionic small-diameter artificial blood vessel prepared in this application (cross-sectional view at the cross-section);

[0030] Figure 2 Schematic diagram of the structure of dividing the mold cavity into small cavities along its width direction with a partition during the preparation operation of this application,

[0031] wherein, 1 - partition board, 2 - cavity;

[0032] Figure 3 Schematic diagram of the two-dimensional collagen film obtained in step (2) of Example 1;

[0033] Figure 4 Morphology diagram of the collagen-based sponge in the middle area of the two-dimensional collagen film obtained in step (2) of Example 1 under an optical microscope;

[0034] Figure 5 Wet-state morphology diagram of the end face of the artificial blood vessel obtained in step (4) of Example 1 under an optical microscope;

[0035] Figure 6 For Figure 5 Magnified wet-state morphology diagram of the local section under an optical microscope;

[0036] Figure 7 Stress-strain curve diagram of the artificial blood vessel obtained in Example 1. SPECIFIC EMBODIMENTS

[0037] A preparation method of a collagen-based bionic small-diameter artificial blood vessel:

[0038] (1) Prepare a high-concentration collagen solution and a low-concentration collagen solution respectively. Through 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS), crosslink each collagen solution respectively.

[0039] The mass concentration of the high-concentration collagen solution is 7.5% - 9.5%, and the mass concentration of the low-concentration collagen solution is 5.5% - 7.0%.

[0040] The crosslinking treatment is as follows: First, disperse 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) in deionized water at a concentration of 15 mg / mL respectively. Then, add the dispersion of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and the dispersion of N-hydroxysuccinimide (NHS) to each collagen solution, and mix and stir at 4°C for 24 h to achieve crosslinking treatment.

[0041] Among them, the mass ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) to collagen is 1:5.0 - 6.5, and the mass ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) to N-hydroxysuccinimide (NHS) is 1 - 1.2:0.8 - 1.2.

[0042] (2) Install a partition to divide the mold cavity with an upward opening into three cavities arranged in sequence. Inject the high-concentration collagen solution after crosslinking treatment in step (1) into the two side cavities respectively, and inject the low-concentration collagen solution after crosslinking treatment in step (1) into the middle cavity. When it is naturally air-dried until the collagen solutions in each cavity are in a non-flowing gel state, remove the partition from the mold cavity. Further, after the collagen gel systems on both sides are fully air-dried into films, perform freeze-drying on the collagen gel system in the middle.

[0043] Among them, the mold cavity is a cuboid cavity with an upward opening, 6 cm long and 4 - 5 cm wide. Two partitions are vertically inserted and arranged in the mold cavity, so as to divide the mold cavity into three cavities along the width direction. The thickness of the partition is 0.23 mm.

[0044] The liquid level height of the low-concentration collagen solution injected into the middle cavity is 1.2 - 2.5 times that of the high-concentration collagen solution injected into any one of the two side cavities. Among them, the liquid level heights of the high-concentration collagen solutions injected into the two side cavities are the same.

[0045] When the collagen gel systems on both sides are fully air-dried into films, 80% - 90% of the water in the collagen gel system in the middle has also evaporated, and it can continue to maintain a non-flowing gel state.

[0046] (3) After demolding the two-dimensional collagen film obtained in step (2), parallel to the base tube along the edge away from its freeze-dried area, and then winding it on the base tube to obtain a three-dimensional multi-layer tube. During the winding process, a high-concentration collagen solution is applied between layers to bond the layers together.

[0047] Among them, the base tube is a rigid plastic catheter with an outer tube diameter (diameter) of 2 - 5 mm.

[0048] (4) After air-drying the three-dimensional multi-layer tube obtained in step (3), the base tube is removed, thus leaving an artificial blood vessel.

[0049] The pure collagen raw materials in the following examples and comparative examples are prepared by extracting from New Zealand bovine Achilles tendons: The fresh bovine Achilles tendons are crushed into granular form, and are defatted and the salt-soluble proteins are removed respectively with an excessive amount of hydrogen peroxide aqueous solution with a mass concentration of 30% and an excessive amount of sodium chloride aqueous solution with a mass concentration of 10%. After pulping and mixing, 5% of pepsin based on the mass of the bovine Achilles tendons is added and enzymatically treated at 4°C for 2 hours. After filtering out the large solid particles, centrifugation is carried out, and the supernatant is subjected to salting out in 1 mol / L sodium chloride aqueous solution. The collagen obtained after sufficient salting out and centrifugation is dissolved in 0.5 mol / L acetic acid aqueous solution, stirred evenly and then transferred to a dialysis bag using distilled water as the dialysis solution for dialysis for three days (changing the distilled water every 24 hours), and the obtained collagen solution is freeze-dried.

[0050] Example 1

[0051] (1) By dissolving pure collagen in 0.5 mol / L Hac aqueous solution, a high-concentration collagen solution with a collagen mass concentration of 8% and a low-concentration collagen solution with a collagen mass concentration of 6% are respectively prepared.

[0052] The carbodiimide (EDC) is dispersed in deionized water at a concentration of 15 mg / mL, and the N-hydroxysuccinimide (NHS) is dispersed in deionized water at a concentration of 15 mg / mL.

[0053] The above-mentioned carbodiimide (EDC) dispersion and N-hydroxysuccinimide (NHS) dispersion are added to the above 8% high-concentration collagen solution according to the mass ratio of carbodiimide (EDC) to collagen of 1:6 and the mass ratio of carbodiimide (EDC) to N-hydroxysuccinimide (NHS) of 1:1, and mixed and stirred at 4°C for 24 h to achieve cross-linking treatment.

[0054] Disperse the above-mentioned carbodiimide (EDC) dispersion and N-hydroxysuccinimide (NHS) dispersion into the 6% low-concentration collagen solution according to the mass ratio of carbodiimide (EDC) to collagen of 1:6 and the mass ratio of carbodiimide (EDC) to N-hydroxysuccinimide (NHS) of 1:1, and mix and stir at 4 °C for 24 h to achieve cross-linking treatment;

[0055] (2) Select a rectangular cavity with an opening length of 6 cm and a width of 4 cm facing upwards as the mold cavity. Insert two partition plates vertically and tightly in the mold cavity to keep it stationary, thereby dividing the mold cavity into three rectangular cavities along the width direction (as shown in the attachment Figure 2 ). The thickness of each partition plate is 0.23 mm. The length of each divided cavity is 6 cm, and the widths along the width direction of the original mold cavity are approximately 1 cm, 1 cm, and 2 cm in sequence,

[0056] Pour 5 mL of the low-concentration collagen solution after cross-linking treatment in step (1) into the middle cavity, pour 3 mL of the high-concentration collagen solution after cross-linking treatment in step (1) into the side cavity with a width of 1 cm, and pour 6 mL of the high-concentration collagen solution after cross-linking treatment in step (1) into the side cavity with a width of 2 cm. When the collagen solutions in each cavity are naturally air-dried to a non-flowing gel state, remove the partition plates. After forming a gel system with a high middle part and low sides, continue to air-dry until the gel systems on both sides are fully dried into films. At this time, the upper surface of the gel system in the middle is 1.5 mm away from the bottom of the mold cavity groove. Place the entire mold cavity in a -80 °C refrigerator for 8 h and then freeze-dry to form a two-dimensional collagen film with collagen-based films on both sides and a collagen-based sponge in the middle, as shown in the attachment Figure 3 : Since the collagen-based sponge in the middle has a certain height compared to the collagen-based films on both sides, a transition zone appears at the junction of the collagen-based sponge and the collagen-based films after drying, which further shows that the collagen-based sponge and the collagen-based films on both sides are integrally connected together,

[0057] Observe the collagen-based sponge in the middle of the above two-dimensional collagen film with an optical microscope, as shown in the attachment Figure 4 ;

[0058] (3) After demolding the two-dimensional collagen film obtained in step (2), align its 6-cm edge parallel to a rigid plastic catheter with an outer diameter (diameter) of 3 mm, and wind it around the plastic catheter. During the winding process, apply a collagen aqueous solution with a mass concentration of 8% between each winding layer;

[0059] (4) After the winding structure obtained in step (3) is naturally air-dried sufficiently, remove the plastic catheter therein, thereby leaving an artificial blood vessel with an inner diameter (diameter) of 3 mm,

[0060] The end face morphology of the artificial blood vessel in the wet state was observed with an optical microscope, as Figure 5 and Figure 6 shown. It can be seen that the wall of the artificial blood vessel is uniform, and there is no gap in the tight connection between layers. The middle layer has a porous structure.

[0061] The artificial blood vessel prepared above was cut into an axial length of 2 cm. After the sample was immersed in 0.01 M PBS buffer solution for 2 hours, it was tested with a biomaterial mechanical property tester (Bose ELF 3200, Bose Corporation, USA). The tensile rate was 5 mm / min, and the stress-strain curve measured is as attached Figure 7 shown,

[0062] wherein, Col-VG represents the overall sample of the artificial blood vessel prepared in Example 1, Col-scaffold represents the sample of the collagen-based sponge prepared in step (2) of Example 1 formed into a tube alone (outer diameter (diameter) of the tube is 3 mm), and Col-film represents the sample of the inner collagen-based film prepared in step (2) of Example 1 formed into a tube alone (outer diameter (diameter) of the tube is 3 mm). It can be seen that the mechanical properties of the artificial blood vessel meet the use requirements and are the best compared.

[0063] Example 2

[0064] (1) By dissolving pure collagen in 0.5 mol / L Hac aqueous solution, a high-concentration collagen solution with a collagen mass concentration of 9% and a low-concentration collagen solution with a collagen mass concentration of 6.5% were respectively prepared.

[0065] The carbodiimide (EDC) was dispersed in deionized water at a concentration of 15 mg / mL, and the N-hydroxysuccinimide (NHS) was dispersed in deionized water at a concentration of 15 mg / mL.

[0066] The above-mentioned carbodiimide (EDC) dispersion and N-hydroxysuccinimide (NHS) dispersion were added to the above 9% high-concentration collagen solution according to the mass ratio of carbodiimide (EDC) to collagen of 1:6 and the mass ratio of carbodiimide (EDC) to N-hydroxysuccinimide (NHS) of 1:1.2, and mixed and stirred at 4°C for 24 h to achieve cross-linking treatment.

[0067] The above-mentioned carbodiimide (EDC) dispersion and N-hydroxysuccinimide (NHS) dispersion were added to the above 6.5% low-concentration collagen solution according to the mass ratio of carbodiimide (EDC) to collagen of 1:6 and the mass ratio of carbodiimide (EDC) to N-hydroxysuccinimide (NHS) of 1:1.2, and mixed and stirred at 4°C for 24 h to achieve cross-linking treatment;

[0068] (2) Select a cuboid cavity with an opening size of 6 cm in length and 4.6 cm in width facing upwards as the mold cavity. Insert two partition plates vertically and tightly in the mold cavity to keep them stationary, thereby dividing the mold cavity into three cuboid cavities along the width direction (as shown in the appendix Figure 2 ). The thickness of each partition plate is 0.23 mm. The length of each divided cavity is 6 cm, and the widths along the width direction of the original mold cavity are approximately 1.3 cm, 1.3 cm, and 2 cm in sequence.

[0069] Pour 6 mL of the low-concentration collagen solution after cross-linking treatment in step (1) into the middle cavity, pour 3 mL of the high-concentration collagen solution after cross-linking treatment in step (1) into the side cavity with a width of 1.3 cm, and pour 4.6 mL of the high-concentration collagen solution after cross-linking treatment in step (1) into the side cavity with a width of 2 cm. When the collagen solutions in each cavity are naturally air-dried to a non-flowing gel state, remove the partition plates. After forming a gel system with a high middle and low sides, continue to air-dry until the gel systems on both sides are fully dried into films. At this time, the upper surface of the middle gel system is 1.2 mm away from the bottom of the mold cavity groove. Place the entire mold cavity in a -80 °C refrigerator for 12 h and then freeze-dry to form a two-dimensional collagen film with collagen-based films on both sides and a collagen-based sponge in the middle.

[0070] (3) After demolding the two-dimensional collagen film obtained in step (2), align its 6-cm edge parallel to a rigid plastic catheter with an outer diameter (diameter) of 4 mm, and wind it around the plastic catheter. During the winding process, apply a 9% (mass concentration) aqueous collagen solution between each winding layer.

[0071] (4) After the winding structure obtained in step (3) is naturally air-dried sufficiently, withdraw the plastic catheter therein, thereby leaving an artificial blood vessel with an inner diameter (diameter) of 4 mm.

[0072] Comparative Example 1

[0073] In step (2), air-dry and dry the collagen in the middle cavity instead of freeze-drying, and the remaining operations are the same as in Example 1:

[0074] (1) By dissolving pure collagen in a 0.5 mol / L aqueous Hac solution, prepare a high-concentration collagen solution with a collagen mass concentration of 8% and a low-concentration collagen solution with a collagen mass concentration of 6% respectively.

[0075] Disperse 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) in deionized water at a concentration of 15 mg / mL, and disperse N-hydroxysuccinimide (NHS) in deionized water at a concentration of 15 mg / mL.

[0076] Disperse the above-mentioned carbodiimide (EDC) dispersion and N-hydroxysuccinimide (NHS) dispersion into the above 8% high-concentration collagen solution according to the mass ratio of carbodiimide (EDC) to collagen of 1:6 and the mass ratio of carbodiimide (EDC) to N-hydroxysuccinimide (NHS) of 1:1, and mix and stir at 4 °C for 24 h to achieve cross-linking treatment.

[0077] Disperse the above-mentioned carbodiimide (EDC) dispersion and N-hydroxysuccinimide (NHS) dispersion into the above 6% low-concentration collagen solution according to the mass ratio of carbodiimide (EDC) to collagen of 1:6 and the mass ratio of carbodiimide (EDC) to N-hydroxysuccinimide (NHS) of 1:1, and mix and stir at 4 °C for 24 h to achieve cross-linking treatment.

[0078] (2) Select a cuboid cavity with an opening of 6 cm in length and 4 cm in width facing upwards as the mold cavity. Insert two partition plates vertically and tightly in the mold cavity to keep them stationary, thereby dividing the mold cavity into three cuboid cavities along the width direction (as shown in the appendix Figure 2 ). The thickness of each partition plate is 0.23 mm. The length of each divided cavity is 6 cm, and the widths are approximately 1 cm, 1 cm, and 2 cm in sequence along the width direction of the original mold cavity.

[0079] Pour 5 mL of the low-concentration collagen solution after cross-linking treatment in step (1) into the middle cavity, pour 3 mL of the high-concentration collagen solution after cross-linking treatment in step (1) into the side cavity with a width of 1 cm, and pour 6 mL of the high-concentration collagen solution after cross-linking treatment in step (1) into the side cavity with a width of 2 cm. When the collagen solutions in each cavity are naturally air-dried to a non-flowing gel state, remove the partition plates. After forming a gel system with a high middle and low sides, continue air-drying until the gel systems in each cavity are fully dried into films, thereby obtaining a two-dimensional collagen film.

[0080] (3) After demolding the two-dimensional collagen film obtained in step (2), align its 6-cm edge parallel to the outer diameter (diameter) of a rigid plastic catheter of 3 mm, and wind it around the plastic catheter. During the winding process, apply a collagen aqueous solution with a mass concentration of 8% between each winding layer.

[0081] (4) After the winding structure obtained in step (3) is naturally air-dried sufficiently, remove the plastic catheter therein, thereby leaving an artificial blood vessel with an inner diameter (diameter) of 3 mm.

[0082] Cell adhesion and growth experiment

[0083] The two-dimensional collagen films prepared in step (2) of the above embodiments and the two-dimensional collagen films prepared in step (2) of the comparative example, while maintaining the film surface orientation in the preparation cavity, are respectively laid flat and immersed in an aqueous ethanol solution with a volume fraction of 70% for 1 hour, then immersed in a 0.01 M PBS buffer solution for 1 hour, irradiated with an ultraviolet lamp for 30 minutes, and then the collagen-based sponges or collagen-based films formed in the middle cavity on each two-dimensional collagen film are respectively cut into several small round pieces with a diameter slightly smaller than the pore diameter of the 96-well plate.

[0084] The small round pieces of the same example sample or comparative example sample are placed in the wells of a 96-well plate in a one-to-one correspondence and laid flat, immersed in α-MEM medium. Human umbilical artery smooth muscle cells are taken, digested with trypsin (0.25%, containing phenol red), and the cell suspension is obtained and the cells are counted. They are inoculated into the above 96-well plate at a cell density of 3×10 3 cells / well, placed in a cell incubator (MCO-18AIC, Sanyo, Japan), cultured in this medium for three days, then the culture medium is discarded, 10 μL of MTT detection solution (5 mg / mL) is added, incubated in an incubator for 4 h and then the culture is terminated, 100 μL of DMSO is added and shaken for 10 min, and the absorbance value (OD value, the larger the OD value, the more cells; in the detection experiment, the sample is basically transparent after soaking for this period of time and has no significant impact on the measurement of absorbance) is measured using a microplate continuous wavelength microplate reader at a wavelength of 450 nm. The results are shown in the following table:

[0085] OD value Example 1 1.8 Example 2 1.5 Comparative Example 1 1.1

[0086] It can be seen that the sponge layer in this solution is more conducive to the growth of human umbilical artery smooth muscle cells compared to the conventional dense collagen film.

Claims

1. A preparation method of a collagen-based bionic small-caliber artificial blood vessel, characterized in that: The preparation method is as follows: (1) Prepare a high-concentration collagen solution and a low-concentration collagen solution respectively. Crosslink each collagen solution through carbodiimide and N-hydroxysuccinimide. Among them, the mass concentration of the high-concentration collagen solution is 7.5% - 9.5%, and the mass concentration of the low-concentration collagen solution is 5.5% - 7.0%; (2) Install a partition to divide the cavity with an upward opening into three cavities arranged in sequence. Inject the high-concentration collagen solution after crosslinking treatment in step (1) into the two side cavities respectively, and inject the low-concentration collagen solution after crosslinking treatment in step (1) into the middle cavity. When the collagen solutions in each cavity are naturally air-dried to a non-flowing gel state, remove the partition from the cavity. Further, after the collagen gel systems on both sides are fully air-dried into films, perform freeze-drying on the collagen gel system in the middle; (3) After demolding the two-dimensional collagen film obtained in step (2), parallel to the base tube along the edge far from its freeze-dried area, and wind it on the base tube to obtain a three-dimensional multi-layer tube. During the winding process, apply a high-concentration collagen solution between layers to make the layers adhere to each other; (4) After air-drying the three-dimensional multi-layer tube obtained in step (3), pull out the base tube to leave the artificial blood vessel; Among them, in step (2), the cavity is a cuboid cavity with an upward opening. The two partitions are vertically inserted and arranged in the cavity to divide the cavity into three cavities along the width direction; The liquid level height of the low-concentration collagen solution injected into the middle cavity is 1.2 - 2.5 times the liquid level height of the high-concentration collagen solution after crosslinking treatment injected into any one of the two side cavities. Among them, the liquid level heights of the high-concentration collagen solutions after crosslinking treatment injected into the two side cavities are the same.

2. The preparation method of the collagen-based bionic small-caliber artificial blood vessel according to claim 1, characterized in that: In step (1), first disperse carbodiimide and N-hydroxysuccinimide in deionized water at a concentration of 15 mg / mL respectively, and then add the obtained carbodiimide dispersion liquid and the obtained N-hydroxysuccinimide dispersion liquid to each collagen solution, and mix and stir at 4°C for 24 h to achieve crosslinking treatment.

3. The preparation method of the collagen-based bionic small-caliber artificial blood vessel according to claim 2, wherein: The mass ratio of carbodiimide to collagen is 1:5.0 - 6.5, and the mass ratio of carbodiimide to N-hydroxysuccinimide is 1 - 1.2:0.8 - 1.

2.

4. The preparation method of the collagen-based bionic small-caliber artificial blood vessel according to claim 1, characterized in that: In step (3), the base tube is a rigid plastic catheter with an outer tube diameter of 2 - 5 mm.

5. An artificial blood vessel prepared by the method according to any one of claims 1 to 4.

6. The artificial blood vessel according to claim 5, wherein: The tube wall of the artificial blood vessel includes a three-layer structure, which are, from the inside to the outside, a first collagen-based film layer, a collagen-based sponge layer, and a second collagen-based film layer.

7. The artificial blood vessel according to claim 6, wherein: The thickness of the collagen-based sponge layer is 1 - 2 mm.

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