Artificial blood vessel basal layer and preparation method thereof

Abstract

The invention discloses an artificial blood vessel basal layer and a preparation method thereof and relates to the technical field of artificial blood vessel preparation. The preparation method of the artificial blood vessel basal layer comprises the following steps: providing a small intestine submucosa subjected to decellularization treatment; the small intestinal submucosa is wound on the surface of a long-strip-shaped base body with the cylindrical outer surface, overlapping areas of the small intestinal submucosa are attached, and then a clamp is used for clamping to obtain a pretreatment piece; the pretreated part is dried and then subjected to heat treatment under the condition that the temperature ranges from 80 DEG C to 150 DEG C, and the overlapped areas of the small intestine submucosa are connected. The small intestine submucosa can be firmly connected, and the problem that embolism is likely to be caused when a micro suture line is adopted for suturing can be solved.

Description

Technical Field

[0001] The present application relates to the technical field of artificial blood vessel preparation, and in particular to an artificial blood vessel basal layer and a preparation method thereof. Background Art

[0002] Artificial blood vessels are substitutes for many severely narrowed or occluded blood vessels. They are mostly artificially made of synthetic materials such as nylon, polyester (Dacron), polytetrafluoroethylene (PTFE), etc., and are suitable for vascular bypass surgery throughout the body. However, these synthetic materials are relatively hard and have poor blood compatibility or biocompatibility, and are not very suitable for the preparation of small-caliber blood vessels (inner diameter ≤ 6mm). The small intestinal submucosa (abbreviated as SIS in English) is a biomaterial with good biocompatibility and mechanical strength. Studies have shown that SIS is more suitable for the preparation of small-caliber artificial blood vessels. However, in existing reports, when the small intestinal submucosa is made into the basal layer of artificial blood vessels, the small intestinal submucosa is generally rolled into a tube and then continuously sutured and fixed with microsutures. However, studies have shown that when sutured with microsutures, the small intestinal submucosa will be exposed to the blood during the degradation process, which is easy to cause embolism. Summary of the invention

[0003] The present application provides an artificial blood vessel basal layer and a preparation method thereof, which can not only make the connection between the submucosal layers of the small intestine more firmly, but also improve the problem of embolism easily caused by suturing with microsutures.

[0004] This application is implemented as follows:

[0005] In a first aspect, the present application provides a method for preparing an artificial blood vessel basal layer, comprising the following steps:

[0006] Providing a decellularized small intestinal submucosa; winding the small intestinal submucosa on a surface of a long strip substrate having a cylindrical outer surface, and making the overlapping areas of the small intestinal submucosa fit together, and then clamping with a clamp to obtain a pre-treated part;

[0007] The pre-treated piece is first dried and then heat treated at a temperature of 80 to 150° C. to connect the overlapping areas of the small intestinal submucosal layer.

[0008] Furthermore, the drying treatment is performed by freeze drying, and the freeze drying step includes: pre-freezing the pre-treated part at a temperature of -80 to -20°C for 4 to 12 hours, and then vacuum drying.

[0009] Furthermore, the heat treatment conditions also include maintaining vacuum while heating. Optionally, the heat treatment time is 1 to 12 hours.

[0010] Furthermore, the step of winding the small intestinal submucosal layer on the surface of a long strip substrate having a cylindrical outer surface comprises:

[0011] Continuously winding a sheet of the small intestinal submucosal layer onto the outer surface of the long strip substrate, so that the outer surface of the long strip substrate has at least two layers of the small intestinal submucosal layer;

[0012] Optionally, the starting and ending positions of the small intestinal submucosal layer are respectively at different positions on the outer peripheral surface of the long strip substrate.

[0013] Furthermore, the step of winding the small intestinal submucosal layer on the surface of a long strip substrate having a circular outer surface comprises:

[0014] The plurality of small intestinal submucosal layers are sequentially wound onto the outer surface of the long strip substrate, so that the beginning and the end of two adjacent small intestinal submucosal layers are staggered and overlapped on the outer peripheral surface of the long strip substrate.

[0015] Furthermore, the staggered regions of the start and end of the plurality of sheets of the small intestinal submucosal layer are located at different positions on the outer peripheral surface of the long strip substrate.

[0016] Furthermore, the overlapping area of ​​the small intestinal submucosal layer has 2 to 10 layers.

[0017] Furthermore, the diameter of the elongated substrate is ≤6 mm.

[0018] Furthermore, the clamp includes a locking mechanism and two clamping members, the clamping member includes a clamping portion and a connecting portion, the clamping portion is provided with a groove for clamping a long strip substrate with the small intestinal submucosal layer wrapped on the outer surface; the groove extends to the two end portions of the clamping member, the connecting portion is provided on the opposite sides of the groove, the longitudinal cross-section of the groove is arc-shaped and the central angle is α, 135°≤α<180°; when the two clamping members clamp the long strip substrate with the small intestinal submucosal layer wrapped on the outer surface, there is a gap between the corresponding connecting portions of the two clamping members; the locking mechanism is configured to connect the connecting portions of the two clamping members, and can make the clamping portion of at least one of the clamping members have a movement tendency to approach the clamping portion of the other clamping member.

[0019] In a second aspect, the present application provides an artificial blood vessel basal layer, which is prepared by the preparation method of the artificial blood vessel basal layer of the first aspect.

[0020] This application has at least the following beneficial effects:

[0021] The preparation method of the artificial blood vessel basal layer of the present application is to wind the decellularized small intestinal submucosal layer on the surface of a long strip substrate, and make the overlapping area fit together. Under the action of a clamp, the layers are tightly fitted together. After drying, heat treatment is performed at 80 to 150°C, so that the tightly fitted layers of the small intestinal submucosal layer react with each other to form a more stable connection structure, and the layers of the artificial blood vessel basal layer formed are not easy to fall off. The preparation method of the present application eliminates the need for suturing with micro sutures, and thus can also avoid the problem of embolism caused by the use of micro sutures. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 An exploded view of a clamp in an embodiment of the present application;

[0023] Figure 2 It is a schematic diagram of the structure of the clamp in the embodiment of the present application;

[0024] Figure 3 It is a structural schematic diagram of another clamp in an embodiment of the present application;

[0025] Figure 4 This is a photograph of the basal layer of the artificial blood vessel prepared in Example 1 of the present application.

[0026] Icons: 10-clamp; 11-clamping piece; 111-clamping portion; 1111-connecting hole; 1112-groove; 1113-polytetrafluoroethylene layer; 112-connecting portion; 1121-connecting hole; 12-locking mechanism; 121-connecting rod; 122-fastener; 1221-nut; 1222-spring; 123-abutment portion; 20-long strip substrate. DETAILED DESCRIPTION

[0027] The embodiments of the present application will be described in detail below in conjunction with the examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present application and should not be considered as limiting the scope of the present application. In the examples, if specific conditions are not specified, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0028] The following is a detailed description of an artificial blood vessel basal layer and a preparation method thereof according to an embodiment of the present application:

[0029] In a first aspect, an embodiment of the present application provides a method for preparing an artificial blood vessel basal layer, comprising the following steps:

[0030] S1: providing a decellularized small intestinal submucosa, winding the small intestinal submucosa on a surface of a long strip substrate having a cylindrical outer surface, and fitting the overlapping areas of the small intestinal submucosa, and then clamping with a clamp 10 to obtain a pre-treated part.

[0031] By winding the decellularized small intestinal submucosal layer on the surface of the long strip substrate and making the overlapping areas fit together, the layers are tightly fitted together under the action of the clamp 10, which is conducive to the subsequent treatment so that the layers of the small intestinal submucosal layer react with each other. Among them, the outer surface of the long strip substrate is cylindrical, and the basal layer of the artificial blood vessel finally formed is a circular tube.

[0032] Exemplarily, if the diameter of the long strip substrate is ≤6 mm, the artificial blood vessel basal layer prepared is suitable for small-caliber artificial blood vessels. Optionally, the overall material of the long strip substrate is polytetrafluoroethylene, or the outer surface of the metal substrate has a polytetrafluoroethylene layer. In addition, the long strip substrate can be a tubular body or a columnar body, solid or hollow, as long as the outer surface is cylindrical. When the surface of the long strip substrate is polytetrafluoroethylene, it is convenient to demold the artificial blood vessel basal layer after subsequent heat treatment.

[0033] In addition, it should be noted that when the small intestinal submucosal layer is wound on the surface of the long strip substrate, one sheet of the small intestinal submucosal layer can be wound on the surface of the long strip substrate, or multiple sheets of the small intestinal submucosal layer can be wound on the surface of the long strip substrate in sequence, which can be selected according to needs.

[0034] In a possible embodiment, the step of winding the small intestinal submucosal layer on the surface of a long strip substrate with a cylindrical outer surface includes: continuously winding a sheet of small intestinal submucosal layer onto the outer surface of the long strip substrate so that the outer surface of the long strip substrate has at least two layers of small intestinal submucosal layer.

[0035] It should be noted that, in this embodiment, making the outer surface of the elongated substrate have at least two layers of small intestinal submucosal layers means that every position on the outer surface of the elongated substrate has at least two layers of small intestinal submucosal layers.

[0036] By continuously winding a sheet of small intestinal submucosal layer onto the outer surface of the long strip substrate, there are fewer joints, and the overall stability of the artificial blood vessel base layer is higher, which is conducive to improving the bursting pressure that the artificial blood vessel can withstand. For example, the starting and ending positions of the small intestinal submucosal layer are respectively at different positions on the outer peripheral surface of the long strip substrate, which means that the starting and ending positions of the small intestinal submucosal layer are staggered on the outer peripheral surface of the long strip substrate, and there are at least three layers overlapping in the thickness direction, which is conducive to improving the bursting pressure that the artificial blood vessel base layer can withstand.

[0037] In another possible embodiment, the step of winding the small intestinal submucosal layer on the surface of a long strip substrate having a cylindrical outer surface comprises:

[0038] Multiple small intestinal submucosal layers are sequentially wound onto the outer surface of the long strip substrate, so that the beginning and end of two adjacent small intestinal submucosal layers are staggered and overlapped on the outer peripheral surface of the long strip substrate. Compared with the method in which the beginning and end of some small intestinal submucosal layers do not cross and overlap, the beginning and end of each small intestinal submucosal layer are arranged in a staggered and overlapping manner, which is more conducive to increasing the bursting pressure that the basal layer of the artificial blood vessel can withstand.

[0039] Furthermore, the staggered regions at the beginning and end of the multiple small intestinal submucosa layers are located at different positions on the outer peripheral surface of the long strip substrate. Since the staggered regions at the beginning and end of each small intestinal submucosa layer are positions that are less susceptible to bursting pressure, setting the staggered regions at the beginning and end of the multiple small intestinal submucosa layers at different positions on the outer peripheral surface of the long strip substrate can disperse the weak positions, which is beneficial to increase the bursting pressure that the artificial blood vessel basal layer can withstand. Optionally, the area range of the staggered regions at the beginning and end of each small intestinal submucosa layer is 1 / 3 to 2 / 3 of the outer peripheral surface of the long strip substrate.

[0040] Exemplarily, the overlapping area of ​​the small intestinal submucosal layer has 2 to 10 layers. It should be noted that the overlapping area in the embodiment of the present application can be the staggered area of ​​the beginning and end of the small intestinal submucosal layer, or the area where the beginning and end of the small intestinal submucosal layer do not form a staggered area.

[0041] In order to ensure that the clamp 10 clamps the long strip substrate 20 with the small intestinal submucosal layer wrapped on the outer surface so that the layers of the small intestinal submucosal layer are tightly fitted, the clamp 10 further includes a locking mechanism 12 and two clamping members 11, the clamping member 11 includes a clamping portion 111 and a connecting portion 112, the clamping portion 111 is provided with a groove 1112 for clamping the long strip substrate 20 with the small intestinal submucosal layer wrapped on the outer surface; the groove 1112 extends to the two ends of the clamping member 11, and the connecting portion 112 is provided in the groove 111 2, the longitudinal cross-section of the groove 1112 is arc-shaped and the central angle is α, 135°≤α<180°; when the two clamping members 11 clamp the long strip substrate 20 with the small intestinal submucosal layer wrapped around the outer surface, there is a gap between the corresponding connecting parts 112 of the two clamping members 11; the locking mechanism 12 is configured to connect the connecting parts 112 of the two clamping members 11, and enable the clamping part 111 of at least one clamping member 11 to have a movement tendency to approach the clamping part 111 of the other clamping member 11.

[0042] Since the longitudinal cross-section of the groove 1112 is an arc, and the central angle α satisfies the condition of 135°≤α<180°, for example, when the radius of the groove 1112 is r, when the two clamping members 11 clamp the elongated substrate (with the outer surface wrapped with the submucosal layer of the small intestine) with a diameter of R (wherein R=2r), there is a gap between the corresponding connecting portions 112 of the two clamping members 11, and the connecting portions 112 of the two clamping members 11 can be connected and fixed by the locking mechanism 12. When the clamping portion 111 of at least one clamping member 11 moves toward the direction approaching the clamping portion 111 of the other clamping member 11, the elongated substrate 20 with the submucosal layer of the small intestine wrapped on the outer surface can be clamped. If the two clamping members 11 clamp the long strip substrate 20 with the small intestinal submucosal layer wrapped around its outer surface, and the corresponding connecting parts 112 of the two clamping members 11 are already attached together with no gap between them, the locking mechanism 12 cannot cause the clamping parts 111 to produce relative movement, which is not conducive to clamping the long strip substrate 20 with the small intestinal submucosal layer wrapped around it.

[0043] Wherein, the material of the clamp 11 is metal, and the metal has a good thermal conductivity, so the heat can be effectively transferred during the heat treatment process, which is conducive to the heat treatment. Optionally, there is a polytetrafluoroethylene layer 1113 in the groove 1112, and the longitudinal section of the polytetrafluoroethylene layer 1113 is an arc ring, and the arc ring can fit the inner wall of the groove 1112. Wherein, the polytetrafluoroethylene layer 1113 can be formed by coating the inner wall of the groove 1112, or it can be a separate structure with the groove 1112, which is a separate polytetrafluoroethylene plate body and can fit the inner wall of the groove 1112. When the clamp 11 clamps the long strip substrate 20 with the small intestinal submucosal layer wrapped around the outer surface, the polytetrafluoroethylene layer 1113 is in direct contact with the small intestinal submucosal layer, which can facilitate the demolding of the small intestinal submucosal layer after heat treatment.

[0044] In addition, the clamping portion 111 is also provided with a connecting hole 1111 connected to the groove 1112, and the connecting hole 1111 extends to the outer surface of the clamping portion 111. The clamping device can be used for freeze-drying of the small intestinal submucosal layer on the outer surface of the long strip substrate 20. During the freeze-drying process, the setting of the connecting hole 1111 is conducive to the discharge of water vapor. When there is a polytetrafluoroethylene layer, the polytetrafluoroethylene layer also has through holes, and the through holes are connected to the connecting holes 1111 in a one-to-one correspondence.

[0045] For further information, please refer to Figure 1-Figure 3In one embodiment, the connection parts 112 of the two clamping members 11 have corresponding connection holes 1121, and the locking mechanism 12 includes a connection rod 121 and a fastener 122. The connection rod 121 can be inserted into the connection hole 1121 corresponding to the connection part 112, and is threadedly connected to the fastener 122 on one side of one of the clamping members 11. The fastener 122 can abut against the clamping member 11, and the end of the connection rod 121 away from the fastener 122 is connected with an abutment part 123 that can abut against the surface of the other clamping member 11. When the abutment part 123 abuts against the surface of the other clamping member 11, by threading the fastener 122 and the connection rod 121, when the fastener 122 continuously abuts against the surface of the connection part 112, the clamping parts 111 of the two clamping members 11 move in a direction close to each other, thereby clamping the long strip substrate with the small intestinal submucosal layer wrapped around the outer surface.

[0046] Optionally, in this embodiment, the connecting hole 1121 is a threaded hole having an internal thread, and the outer wall of the connecting rod 121 is provided with an external thread. The external thread of the connecting rod 121 can cooperate with the internal thread of the connecting hole 1121. When the connecting rod 121 passes through the connecting hole 1121, the threaded cooperation makes it difficult for the connecting rod 121 to fall out of the connecting hole 1121, thereby facilitating the threaded connection between the connecting rod 121 and the fastener 122.

[0047] Please refer to Figure 3 Exemplarily, the fastener 122 includes a nut 1221 and a spring 1222. The spring 1222 is sleeved on the connecting rod 121, and the two ends are respectively against the nut 1221 and the clamping member 11. The nut 1221 can be threadedly connected with the connecting rod 121. The outer wall of the connecting rod 121 has an external thread, and the threaded connection can be achieved by matching the external thread of the nut 1221 and the connecting rod 121. Since the spring 1222 can be stretched and compressed, when the nut 1221 moves in a direction close to the spring 1222, the spring 1222 is compressed and generates a restoring force acting on the nut 1221 and the connecting portion 112, so that it can be more conducive to clamping the long strip substrate with the small intestinal submucosal layer wrapped around the outer surface.

[0048] It should be noted that in other embodiments, clamps 10 of other structures may also be used, as long as they can clamp the long strip substrate with the small intestinal submucosal layer wrapped around its surface.

[0049] S2: The pre-treated piece is first dried and then heat-treated at a temperature of 80 to 150° C. to connect the overlapping areas of the small intestinal submucosal layer.

[0050] The inventors of the present application found in the experiment that the pre-treatment parts are clamped so that the layers of the small intestinal submucosal layer are closely attached to each other, and then dried to reduce the moisture content of the small intestinal submucosal layer, making the small intestinal submucosal layer more loose and porous, and then subjected to heat treatment at a temperature of 80 to 150°C, the layers of the small intestinal submucosal layer can easily react and connect to form a three-dimensional network structure, thereby connecting and fixing together, and the basal layer of the artificial blood vessel formed can withstand a large bursting pressure. The inventors of the present application found in the study that too high or too low a treatment temperature affects the mechanical properties of the basal layer of the artificial blood vessel. Optionally, the temperature of the heat treatment is any one of 80°C, 90°C, 100°C, 110°C, 120°C, 130°C, 140°C and 150°C, or a value between any two of them.

[0051] In addition, the heat treatment conditions also include maintaining a vacuum while heating, which can isolate foreign matter or air from entering to prevent other reactions. Moreover, it is easier to remove water vapor and reduce the moisture in the submucosal layer of the small intestine, which is more conducive to reactions between layers to form a three-dimensional network structure and a tighter connection.

[0052] Exemplarily, the heat treatment time is 1 to 12 hours. If the heat treatment time is too short, the reaction degree between the layers of the small intestinal submucosal layer will be low, and the connection degree will be low, resulting in a low bursting pressure that the basal layer of the manufactured artificial blood vessel can withstand; since the main component of the small intestinal submucosal layer contains collagen fibers, if the heat treatment time is too long, it may cause the collagen fiber structure to be destroyed, thereby reducing the tension strength of the basal layer of the manufactured artificial blood vessel. Optionally, the heat treatment time is 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11h and 12h, or a value between any two of them.

[0053] Furthermore, the drying treatment is carried out by freeze drying, and the freeze drying step includes: pre-freezing the pre-treated part at a temperature of -80 to -20°C for 4 to 12 hours, and then vacuum drying.

[0054] By pre-freezing the pretreated parts at a temperature of -80 to -20°C for 4 to 12 hours, the moisture in the submucosal layer of the small intestine can be formed into ice crystals, and then vacuum drying is performed to make the ice crystals sublime into water vapor, so that the moisture in the submucosal layer of the small intestine is fully removed. Moreover, pre-freezing and then vacuum drying can form a porous structure in the submucosal layer of the small intestine. During heat treatment, the porous structure is more conducive to making the layers tightly connected than a smooth surface.

[0055] Exemplarily, the pre-freezing temperature is any one of -80°C, -70°C, -60°C, -50°C, -40°C, -30°C and -20°C or a value between any two thereof. Exemplarily, the pre-freezing time is 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11h or 12h.

[0056] In a second aspect, the present application provides an artificial blood vessel basal layer, which is prepared by the preparation method of the artificial blood vessel basal layer of the present application.

[0057] The preparation method of the present application can not only make the connection between the layers of the small intestinal submucosal layer more firmly, improve the bursting pressure and tension strength of the basal layer of the artificial blood vessel, but also improve the problem of embolism easily caused by suturing with microsutures.

[0058] The artificial blood vessel basal layer and the preparation method thereof of the present application are further described in detail below in conjunction with the examples.

[0059] Example 1

[0060] This embodiment provides a method for preparing an artificial blood vessel basal layer, which comprises the following steps:

[0061] (1) The decellularized small intestinal submucosa is provided. HE staining of the decellularized small intestinal submucosa shows no cell nuclei.

[0062] (2) A polytetrafluoroethylene rod is provided as a long strip substrate, the outer surface of which is cylindrical, with a diameter of 4 mm and a length of 10 cm; the small intestinal submucosal layer with a width of 5.6 cm and a length of 6 cm in step (1) is wound on the outer surface of the polytetrafluoroethylene rod, and the overlapping areas of the small intestinal submucosal layer are made to fit together, and then clamped with a clamp 10 (such as Figure 2 The pre-treated part is obtained as shown in the figure; wherein the length direction of the small intestinal submucosal layer is consistent with the length direction of the long strip matrix.

[0063] (3) The pre-treated piece obtained in step (2) is first pre-frozen at -80°C for 4 hours and then vacuum dried, then heat treated at 80°C for 12 hours, taken out and cooled to room temperature, and then the clamp is removed to obtain an artificial blood vessel basal layer. The obtained artificial blood vessel basal layer is as follows Figure 4 shown.

[0064] Example 2

[0065] This embodiment provides a method for preparing an artificial blood vessel basal layer, which comprises the following steps:

[0066] (1) The decellularized small intestinal submucosa is provided. HE staining of the decellularized small intestinal submucosa shows no cell nuclei.

[0067] (2) A polytetrafluoroethylene rod is provided as a long strip substrate, the outer surface of which is cylindrical, with a diameter of 4 mm and a length of 10 cm; the small intestinal submucosal layer with a width of 5.6 cm and a length of 6 cm in step (1) is wound on the outer surface of the polytetrafluoroethylene rod, and the overlapping areas of the small intestinal submucosal layer are made to fit together, and then clamped with a clamp 10 (such as Figure 2 The pre-treated part is obtained as shown in the figure; wherein the length direction of the small intestinal submucosal layer is consistent with the length direction of the long strip matrix.

[0068] (3) The pretreated piece obtained in step (2) is first pre-frozen at -80°C for 4 hours and then vacuum dried, and then heat treated at 100°C for 12 hours. After cooling to room temperature, the clamp is removed to obtain the artificial blood vessel basal layer.

[0069] Example 3

[0070] This embodiment provides a method for preparing an artificial blood vessel basal layer, which comprises the following steps:

[0071] (1) The decellularized small intestinal submucosa is provided. HE staining of the decellularized small intestinal submucosa shows no cell nuclei.

[0072] (2) providing a polytetrafluoroethylene rod as a long strip substrate, the outer surface of which is circular, the diameter is 4 mm, and the length is 10 cm; winding the small intestinal submucosal layer with a width of 5.6 cm and a length of 6 cm on the outer surface of the polytetrafluoroethylene rod, and making the overlapping areas of the small intestinal submucosal layer fit together, and then clamping it with a clamp 10 (such as Figure 2 The pre-treated part is obtained as shown in the figure; wherein the length direction of the small intestinal submucosal layer is consistent with the length direction of the long strip matrix.

[0073] (3) The pretreated piece obtained in step (2) is first pre-frozen at -80°C for 4 hours and then vacuum dried, and then heat treated at 120°C for 12 hours. After cooling to room temperature, the clamp is removed to obtain the artificial blood vessel basal layer.

[0074] Example 4

[0075] This embodiment provides a method for preparing an artificial blood vessel basal layer, which comprises the following steps:

[0076] (1) The decellularized small intestinal submucosa is provided. HE staining of the decellularized small intestinal submucosa shows no cell nuclei.

[0077] (2) providing a polytetrafluoroethylene rod as a long strip substrate, the outer surface of which is circular, the diameter is 4 mm, and the length is 10 cm; winding the small intestinal submucosal layer with a width of 5.6 cm and a length of 6 cm on the outer surface of the polytetrafluoroethylene rod, and making the overlapping areas of the small intestinal submucosal layer fit together, and then clamping it with a clamp 10 (such as Figure 2 The pre-treated part is obtained as shown in the figure; wherein the length direction of the small intestinal submucosal layer is consistent with the length direction of the long strip matrix.

[0078] (3) The pretreated piece obtained in step (2) is first pre-frozen at -80°C for 4 hours and then vacuum dried, and then heat treated at 150°C for 12 hours. After cooling to room temperature, the clamp is removed to obtain the artificial blood vessel basal layer.

[0079] Example 5

[0080] This embodiment provides a method for preparing an artificial blood vessel basal layer, which comprises the following steps:

[0081] (1) The decellularized small intestinal submucosa is provided. HE staining of the decellularized small intestinal submucosa shows no cell nuclei.

[0082] (2) providing a polytetrafluoroethylene rod as a long strip substrate, the outer surface of which is circular, the diameter is 4 mm, and the length is 10 cm; winding two sheets of small intestinal submucosal layers with a width of 2.8 cm and a length of 6 cm in step (1) on the outer surface of the polytetrafluoroethylene rod in sequence, and making the overlapping areas of the small intestinal submucosal layers fit together; wherein the starting position of the second sheet of small intestinal submucosal layer is aligned with the end position of the first sheet of small intestinal submucosal layer; and then clamping with a clamp 10 (such as Figure 2 The pre-treated part is obtained as shown in the figure; wherein the length direction of the small intestinal submucosal layer is consistent with the length direction of the long strip matrix.

[0083] (3) The pretreated piece obtained in step (2) is first pre-frozen at -80°C for 4 hours and then vacuum dried, and then heat treated at 150°C for 3 hours. After cooling to room temperature, the clamp is removed to obtain the artificial blood vessel basal layer.

[0084] Example 6

[0085] This embodiment provides a method for preparing an artificial blood vessel basal layer, which comprises the following steps:

[0086] (1) The decellularized small intestinal submucosa is provided. HE staining of the decellularized small intestinal submucosa shows no cell nuclei.

[0087] (2) providing a polytetrafluoroethylene rod as a long strip substrate, the outer surface of which is circular, the diameter is 4 mm, and the length is 10 cm; winding two small intestinal submucosal layers with a width of 3 cm and a length of 6 cm in step (1) on the outer surface of the polytetrafluoroethylene rod in sequence, and making the overlapping areas of the small intestinal submucosal layers fit together; wherein the starting position of the second small intestinal submucosal layer is staggered and overlapped with the end of the first small intestinal submucosal layer, and the width of the overlapping area is about 4 mm; and then clamping with a clamp 10 (such as Figure 2 The pre-treated part is obtained as shown in the figure; wherein the length direction of the small intestinal submucosal layer is consistent with the length direction of the long strip matrix.

[0088] (3) The pretreated piece obtained in step (2) is first pre-frozen at -80°C for 4 hours and then vacuum dried, and then heat treated at 150°C for 3 hours. After cooling to room temperature, the clamp is removed to obtain the artificial blood vessel basal layer.

[0089] Example 7

[0090] This embodiment provides a method for preparing an artificial blood vessel basal layer, which comprises the following steps:

[0091] (1) The decellularized small intestinal submucosa is provided. HE staining of the decellularized small intestinal submucosa shows no cell nuclei.

[0092] (2) providing a polytetrafluoroethylene rod as a long strip substrate, the outer surface of which is circular, the diameter is 4 mm, and the length is 10 cm; winding the small intestinal submucosal layer with a width of 5.6 cm and a length of 6 cm on the outer surface of the polytetrafluoroethylene rod, and making the overlapping areas of the small intestinal submucosal layer fit together, and then clamping it with a clamp 10 (such as Figure 2 The pre-treated part is obtained as shown in the figure; wherein the length direction of the small intestinal submucosal layer is consistent with the length direction of the long strip matrix.

[0093] (3) The pretreated piece obtained in step (2) is first pre-frozen at -40°C for 8 hours and then vacuum dried, and then heat treated at 120°C for 6 hours. After cooling to room temperature, the clamp is removed to obtain the artificial blood vessel basal layer.

[0094] Example 8

[0095] This embodiment provides a method for preparing an artificial blood vessel basal layer. Compared with Embodiment 1, the only difference is that in this embodiment, a vacuum state is maintained while performing heat treatment.

[0096] Comparative Example 1

[0097] Comparative Example 1 provides a method for preparing an artificial blood vessel basal layer. Compared with Example 1, the only difference is that the heat treatment temperature in Comparative Example 1 is 60°C.

[0098] Comparative Example 2

[0099] Comparative Example 1 provides a method for preparing an artificial blood vessel basal layer. Compared with Example 1, the only difference is that the heat treatment temperature in Comparative Example 2 is 170°C.

[0100] Comparative Example 3

[0101] Comparative Example 3 provides a method for preparing an artificial blood vessel basal layer. Compared with Example 1, Comparative Example 3 omits the step of pre-freezing at -80°C for 4 hours and then vacuum drying.

[0102] Test example

[0103] One end of the artificial blood vessel basal layer prepared in Examples 1 to 4, Example 8 and Comparative Examples 1 to 3 was respectively inserted into a hose and fixed, the other end of the artificial blood vessel basal layer was sealed, and the other end of the hose was connected to a pressure detection device and a water injection device. During the test, physiological saline was injected into the artificial blood vessel basal layer at a rate of 2 mL / min through the water injection device, and the pressure when the side wall of the artificial blood vessel basal layer ruptured and water seeped was recorded, which was recorded as the bursting strength. The bursting strengths of the embodiments and comparative examples are shown in Table 1.

[0104] Table 1. Burst strength test results

[0105]

[0106]

[0107] From the results in Table 1, it can be seen that the bursting strength of the artificial blood vessel basal layer of Examples 1 to 4 and Example 8 is greater than 45 KPa, while the bursting strength of the artificial blood vessel basal layer of Comparative Examples 1 to 3 is less than that of the examples. This indicates that the preparation method of the artificial blood vessel basal layer of the embodiment of the present application can obtain an artificial blood vessel basal layer with higher bursting strength.

[0108] Among them, comparing Examples 1 to 4, Comparative Example 1 and Comparative Example 2, the heat treatment temperatures of Comparative Example 1 and Comparative Example 2 are not within the range specified in the present application, and the bursting strengths of the artificial blood vessel basal layers of Comparative Example 1 and Comparative Example 2 are both less than 36 KPa, and the bursting strengths of the artificial blood vessel basal layers of Examples 1 to 4 are both greater than 45 KPa, indicating that the heat treatment temperature is limited to the range of the present application to obtain an artificial blood vessel basal layer with higher bursting strength.

[0109] By comparing Example 1 and Comparative Example 3, Comparative Example 3 omits the step of pre-freezing for 4 hours at a temperature of -80°C and then vacuum drying. The results show that the bursting strength of the basal layer of the artificial blood vessel in Comparative Example 3 is much smaller than the bursting strength of the basal layer of the artificial blood vessel in Example 1, indicating that the bursting strength of the basal layer of the artificial blood vessel can be effectively improved by freezing, vacuum drying and then heat treatment.

[0110] By comparing Example 1 and Example 8, Example 8 maintained a vacuum state while performing heat treatment, and the bursting strength of the artificial blood vessel basal layer prepared in Example 8 was greater than the bursting strength of the artificial blood vessel basal layer in Example 1, indicating that maintaining a vacuum state while performing heat treatment is conducive to obtaining an artificial blood vessel basal layer with higher bursting strength.

[0111] The above are only specific embodiments of the present application and are not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing an artificial blood vessel basal layer, characterized in that: The following steps are involved: Providing a decellularized small intestinal submucosa; winding the small intestinal submucosa on a surface of a long strip substrate having a cylindrical outer surface, and making the overlapping areas of the small intestinal submucosa fit together, and then clamping with a clamp to obtain a pre-treated part; The pre-treated piece is first dried and then heat treated at a temperature of 80 to 150° C. to connect the overlapping areas of the small intestinal submucosal layer.

2. The method for preparing the artificial blood vessel basal layer according to claim 1, characterized in that: The drying treatment is carried out in a freeze-drying manner, and the freeze-drying step comprises: pre-freezing the pre-treated part at a temperature of -80 to -20°C for 4 to 12 hours, and then vacuum drying.

3. The method for preparing the artificial blood vessel basal layer according to claim 1, characterized in that: The heat treatment conditions further include maintaining vacuum while heating; optionally, the heat treatment time is 1 to 12 hours.

4. The method for preparing an artificial blood vessel basal layer according to any one of claims 1 to 3, characterized in that: The step of winding the small intestinal submucosal layer on the surface of a long strip substrate having a cylindrical outer surface comprises: Continuously winding a sheet of the small intestinal submucosal layer onto the outer surface of the long strip substrate, so that the outer surface of the long strip substrate has at least two layers of the small intestinal submucosal layer; Optionally, the starting and ending positions of the small intestinal submucosal layer are respectively at different positions on the outer peripheral surface of the long strip substrate.

5. The method for preparing an artificial blood vessel basal layer according to any one of claims 1 to 3, characterized in that: The step of winding the small intestinal submucosal layer on the surface of a long strip substrate having a cylindrical outer surface comprises: The plurality of small intestinal submucosal layers are sequentially wound onto the outer surface of the long strip substrate, so that the beginning and the end of two adjacent small intestinal submucosal layers are staggered and overlapped on the outer peripheral surface of the long strip substrate.

6. The method for preparing the artificial blood vessel basal layer according to claim 5, characterized in that: The starting and ending interlaced areas of the plurality of small intestinal submucosal layers are located at different positions on the outer peripheral surface of the long strip substrate.

7. The method for preparing an artificial blood vessel basal layer according to any one of claims 1 to 3, characterized in that: The overlapping area of ​​the small intestinal submucosal layer has 2 to 10 layers.

8. The method for preparing an artificial blood vessel basal layer according to any one of claims 1 to 3, characterized in that: The diameter of the long strip substrate is ≤6 mm.

9. The method for preparing an artificial blood vessel basal layer according to any one of claims 1 to 3, characterized in that: The clamp includes a locking mechanism and two clamping members, the clamping member includes a clamping portion and a connecting portion, the clamping portion is provided with a groove for clamping a long strip substrate with the small intestinal submucosal layer wrapped on the outer surface; the groove extends to the two end portions of the clamping member, and the connecting portion is arranged on the opposite sides of the groove, the longitudinal cross-section of the groove is arc-shaped and the central angle is α, 135°≤α<180°; when the two clamping members clamp the long strip substrate with the small intestinal submucosal layer wrapped on the outer surface, there is a gap between the corresponding connecting portions of the two clamping members; the locking mechanism is configured to connect the connecting portions of the two clamping members, and can make the clamping portion of at least one of the clamping members have a movement tendency to approach the clamping portion of the other clamping member.

10. An artificial blood vessel basal layer, characterized in that: The artificial blood vessel is prepared by the preparation method of the artificial blood vessel basal layer according to any one of claims 1 to 9.

Images