An artificial blood vessel with gradient inner and outer apertures, and a preparation method and application thereof

Abstract

The application provides a preparation method of an artificial blood vessel with gradient inner and outer apertures. The application realizes the preparation of the artificial blood vessel with the outer aperture suitable for tissue growth and the inner aperture integrated and dense by electrostatic spinning through two kinds of matrix solutions with different concentrations and different flow rate parameters, so that the time cost and the economic cost are greatly saved. The preparation method of the artificial blood vessel provided by the application is simple, and the demand for the artificial blood vessel can be effectively met. After the artificial blood vessel prepared by the application is implanted into the body, the outer layer of the artificial blood vessel has the aperture suitable for tissue growth, which is beneficial to the adhesion and growth of cells, the inner layer has the dense aperture, which prevents the occurrence of blood leakage, and the good performance meets the use of the artificial blood vessel.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to an artificial blood vessel with gradient inner and outer pore diameters, a preparation method thereof and application. BACKGROUND

[0002] Cardiovascular disease (CVD) is a major cause of morbidity and mortality worldwide, affecting about 523 million people. The most common cause of cardiovascular disease is atherosclerosis, i.e. the accumulation of fatty plaques in the arterial wall, leading to arterial obstruction and reduced blood flow to downstream tissues.

[0003] At present, autologous blood vessel transplantation is the most ideal treatment in clinic, and the patient's autologous artery is very effective in the long term after transplantation. However, due to the limited source and donor site lesions, its use is limited. Therefore, in most cases, artificial blood vessels need to be used for blood vessel transplantation, but the current artificial blood vessels represented by polyethylene terephthalate and expanded polytetrafluoroethylene have been applied in large-diameter blood vessels. However, the current products do not have regenerative capacity and do not have controllable pore size characteristics, and cannot achieve the regeneration and protection of blood vessels. There is always intimal hyperplasia and coagulation risk. It is worth noting that small-diameter artificial blood vessels are more prone to thrombosis, intimal hyperplasia and other problems after being implanted into the body. At present, there is no small-diameter artificial blood vessel (diameter < 6 mm) product that can be applied to heart bypass, knee below peripheral blood vessel replacement and other surgeries.

[0004] There are many studies on multi-layer small-diameter artificial blood vessels at present. The multi-layer artificial blood vessels in the existing research are mostly made by simply stacking different materials layer by layer. The inner layer has a dense pore diameter, which can prevent blood leakage and coagulation; the outer layer has a pore size suitable for tissue growth, which is beneficial to promote tissue and endothelial regeneration after artificial blood vessel implantation. The inner and outer layers are prepared by spinning separately to prepare artificial blood vessels. The inner and outer layers change the pore size and filament diameter through simple stacking, but the pore size and filament diameter between the different layers of artificial blood vessels prepared by this technology do not gradually change. Directly through simple stacking, the bonding force between the inner and outer layers is insufficient, and delamination is easy to occur.

[0005] Most of the multi-layer artificial blood vessels in the existing research are prepared by simple layer-by-layer stacking process using different materials or different structures, such as an inner layer prepared into a structure with small pore diameter and dense structure, and an outer layer prepared into a structure with large pore diameter and loose structure. However, the artificial blood vessels prepared by this technology use a multi-step process, and the prepared blood vessels are not a continuous whole. Even if various bonding processes are used, delamination is still easy to occur.

[0006] Therefore, it is a problem to be solved to provide an artificial blood vessel with a tightly combined structure and different inner and outer layer pore diameters. SUMMARY

[0007] Therefore, the present application aims to provide a kind of artificial blood vessel with gradient inner and outer aperture and its preparation method and application, the artificial blood vessel provided by the present application is integrated structure, structure is closely combined and can reach the effect of different aperture of inner and outer layer.

[0008] The present application provides a kind of preparation method of artificial blood vessel with gradient inner and outer aperture, comprising the following steps:

[0009] A) respectively prepare first matrix solution and second matrix solution with different concentrations;

[0010] The first matrix solution and the second matrix solution include high molecular material;

[0011] The concentration of the first matrix solution is less than the concentration of the second matrix solution;

[0012] B) the first matrix solution and the second matrix solution are mixed after running in respective flow mode, to obtain mixed matrix solution;

[0013] The flow mode of the first matrix solution is: with first initial flow rate and combined deceleration gradually reduces flow rate;

[0014] The flow mode of the second matrix solution is: with second initial flow rate and combined acceleration gradually increases flow rate;

[0015] The first initial flow rate is higher than the second initial flow rate;

[0016] C) the mixed matrix solution is electrospun on the surface of the receiver, to obtain integrated artificial blood vessel.

[0017] Preferably, the high molecular material includes at least one of polycaprolactone, polylactic acid, polyurethane, poly (lactic-co-glycolic acid), poly (L-lactide-co-caprolactone), poly (p-dioxanone) or any ratio mixture of several thereof.

[0018] Preferably, the solvent used for preparing the first matrix solution and the second matrix solution is selected from at least one of hexafluoroisopropanol, chloroform, methanol or any ratio mixture of several thereof.

[0019] Preferably, the concentration of the first matrix solution is 0.1-0.3 g / mL;

[0020] The concentration of the second matrix solution is 0.2-0.5 g / mL.

[0021] Preferably, the first initial flow rate is 10-12 mL / h, and the deceleration is-144--720 mL / h 2 ;

[0022] The second initial flow rate is 0-2 mL / h, and the acceleration is 144-720 mL / h. 2 .

[0023] Preferably, the high-voltage direct current voltage of the electrospinning is 10-12 kV, the distance from the receiver is 10-15 cm, and the spinning time is 10-12 min.

[0024] Preferably, the receiver is a grounded stainless steel rod with a diameter of 2-8 mm.

[0025] The rotation speed of the receiver is 120-180 rpm.

[0026] The application also provides an artificial blood vessel with gradient inner and outer apertures prepared by the preparation method.

[0027] The application also provides an application of the artificial blood vessel in the field of tissue engineering.

[0028] Preferably, the artificial blood vessel is used for vascular patch, valvular blood vessel, and heart valve.

[0029] Compared with the prior art, the application provides a preparation method of an artificial blood vessel with gradient inner and outer apertures, which comprises the following steps: A) preparing first and second matrix solutions with different concentrations, respectively; the first and second matrix solutions comprise a high polymer material; the concentration of the first matrix solution is lower than that of the second matrix solution; B) mixing the first and second matrix solutions after running in respective flow modes to obtain a mixed matrix solution; the flow mode of the first matrix solution is running at a first initial flow rate and gradually reducing the flow rate in combination with a deceleration; the flow mode of the second matrix solution is running at a second initial flow rate and gradually increasing the flow rate in combination with an acceleration; the first initial flow rate is higher than the second initial flow rate; C) electrospinning the mixed matrix solution on the surface of a receiver to obtain an integrated artificial blood vessel. The application realizes the preparation of an artificial blood vessel with an outer layer aperture suitable for tissue growth and an inner layer aperture for integrated densification by electrospinning of two matrix solutions with different concentrations and different flow rate parameters, which greatly saves the time cost and economic cost. The preparation method of the artificial blood vessel provided by the application is simple and can effectively meet the demand for artificial blood vessels. After being implanted into the body, the artificial blood vessel has an outer layer with an aperture suitable for tissue growth, which is conducive to cell adhesion and growth, and an inner layer with a dense aperture that prevents blood leakage, thereby meeting the use of the artificial blood vessel. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 The figure is a schematic diagram of the preparation method of the artificial blood vessel with gradient inner and outer apertures provided by the application.

[0031] Figure 2 The light absorption value of the hole plate at 490nm is detected by a multifunctional enzyme label instrument to represent the adhesion data of the platelets;

[0032] Figure 3 The light absorption value of the hole plate at 450nm is detected by a multifunctional enzyme label instrument to represent the activation data of the platelets. DETAILED DESCRIPTION

[0033] The application provides a preparation method of an artificial blood vessel with gradient inner and outer diameters, comprising the following steps:

[0034] A) respectively preparing a first matrix solution and a second matrix solution with different concentrations;

[0035] The first matrix solution and the second matrix solution comprise a high polymer material;

[0036] The concentration of the first matrix solution is less than that of the second matrix solution;

[0037] B) mixing the first matrix solution and the second matrix solution after running in respective flow modes to obtain a mixed matrix solution;

[0038] The flow mode of the first matrix solution is running at a first initial flow rate and gradually reducing the flow rate in combination with a deceleration;

[0039] The flow mode of the second matrix solution is running at a second initial flow rate and gradually increasing the flow rate in combination with an acceleration;

[0040] The first initial flow rate is higher than the second initial flow rate;

[0041] C) electrospinning the mixed matrix solution on a receiver surface to obtain an integrated artificial blood vessel.

[0042] The application first respectively prepares a first matrix solution and a second matrix solution with different concentrations;

[0043] The first matrix solution and the second matrix solution comprise a high polymer material;

[0044] The concentration of the first matrix solution is less than that of the second matrix solution;

[0045] In the application, the high polymer material comprises at least one or several arbitrary proportion mixtures of polycaprolactone, polylactic acid, polyurethane, poly-lactic-glycolic acid copolymer, poly-L-lactide-caprolactone and poly-p-dioxanone, and is preferably polycaprolactone.

[0046] The solvent used for preparing the first and second matrix solutions is selected from at least one of hexafluoroisopropanol, chloroform, methanol or a mixture of any proportion of several thereof, preferably a mixed solution of chloroform and methanol, and more preferably, the volume ratio of the chloroform and methanol is 1:1 to 10:1, preferably 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, or any value between 1:1 and 10:1.

[0047] In the present application, the concentration of the first matrix solution is 0.1 to 0.3 g / mL, which can be 0.1, 0.15, 0.2, 0.25, 0.3, or any value between 0.1 and 0.3 g / mL.

[0048] The concentration of the second matrix solution is 0.2 to 0.5 g / mL, which can be 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, or any value between 0.2 and 0.5 g / mL.

[0049] After the two matrix solutions are prepared, the two matrix solutions are mixed after running in their respective flow modes to obtain a mixed matrix solution.

[0050] Specifically, the flow mode of the first matrix solution is to run at a first initial flow rate and gradually reduce the flow rate in combination with a deceleration.

[0051] The flow mode of the second matrix solution is to run at a second initial flow rate and gradually increase the flow rate in combination with an acceleration.

[0052] The first initial flow rate is higher than the second initial flow rate.

[0053] In some embodiments of the present application, the first initial flow rate is 10 to 12 mL / h, which can be 10, 10.5, 11, 11.5, 12, or any value between 10 and 12 mL / h, and the deceleration is -144 to -720 mL / h 2 , which can be -144, -150, -200, -250, -300, -350, -400, -450, -500, -550, -600, -650, -700, -720, or any value between -144 and -720 mL / h. 2 The deceleration is 0 to 2 mL / h to stop running, which can be 0, 0.5, 1, 1.5, 2, or any value between 0 and 2 mL / h.

[0054] The second initial flow rate is 0 to 2 mL / h, which can be 0, 0.5, 1, 1.5, 2, or any value between 0 and 2 mL / h, and the acceleration is 144 to 720 mL / h 2Any value between 144, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 720, or 144-720 mL / h, accelerate to 10-12 mL / h stop running, any value between 10, 10.5, 11, 11.5, 12, or 10-12 mL / h. 2 Any value between 144, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 720, or 144-720 mL / h, accelerate to 10-12 mL / h stop running, any value between 10, 10.5, 11, 11.5, 12, or 10-12 mL / h.

[0055] In some embodiments of the present application, the absolute value of the deceleration is greater than or equal to the value of the acceleration.

[0056] In some embodiments of the present application, the final speed of the first matrix solution is 0-2 mL / h, and the final speed of the second matrix solution is 10-12 mL / h.

[0057] In the present application, the acceleration is calculated according to the formula I:

[0058] a=(V a -V b ) / (h a -h b ) Formula I;

[0059] In formula I, V a represents the speed at time a, V b represents the speed at time b, and the unit is mL / h;

[0060] h a represents the time at time a, h b represents the time at time b, and the unit is h;

[0061] The unit of acceleration a is mL / h 2 .

[0062] Similarly, the deceleration is also calculated according to the formula I, and the calculated value is negative.

[0063] The concentration of the first matrix solution and the concentration of the second matrix solution determine the minimum pore size and the maximum pore size of the artificial blood vessel; the matrix solution of the artificial blood vessel at the beginning is mainly the first matrix solution, then the second matrix solution is increased and the first matrix solution is reduced, and the concentration of the mixed solution is continuously increased, so that the artificial blood vessel achieves the effect of uniform increase of pore size and wire diameter.

[0064] In some preferred embodiments of the present application, the method of mixing the first matrix solution and the second matrix solution after running in the respective flow mode is as follows:

[0065] The first matrix solution is loaded into syringe 1, and the second matrix solution is loaded into syringe 2. After inverting the syringes to remove air bubbles, a 21G needle is installed and secured. The syringe is then used to advance the solution using a syringe pump, with both syringes advancing simultaneously. The initial flow rate of syringe 2 is set to 0-2 mL / h, and then increased to 144-720 mL / h. 2 The acceleration increases the flow rate of the solution; the initial flow rate of another syringe 1 is set to 10⁻¹² mL / h, with a flow rate of -144 to -720 mL / h. 2 The deceleration reduces the flow rate of the solution. The solutions from the two syringes are then mixed and electrospinned. This invention does not impose any particular limitation on the mixing method; preferably, the solutions from the two syringes can be mixed using a needle adapter.

[0066] In this invention, the high voltage DC voltage for electrospinning is 10~12kV, which can be any value between 10, 10.5, 11, 11.5, 12, or 10~12kV; the distance to the receiver is 10~15cm, which can be any value between 10, 11, 12, 13, 14, 15, or 10~15cm; and the spinning time is 10~12min, which can be any value between 10, 11, 12, or 10~12min.

[0067] In some specific embodiments of the present invention, the receiver is a grounded stainless steel rod with a diameter of 2 to 8 mm, which can be 2, 3, 4, 5, 6, 7, 8, or any value between 2 and 8 mm.

[0068] The receiver's rotation speed is 120~180 rpm, and can be any value between 120, 130, 140, 150, 160, 170, 180, or 120~180 rpm.

[0069] See Figure 1 , Figure 1 This is a schematic diagram illustrating the method for preparing an artificial blood vessel with gradient inner and outer pore sizes provided by the present invention.

[0070] This invention employs an integrated tube-forming method. By adjusting the speed of the blended matrix syringe, the pore size and filament diameter between each layer are increased uniformly. This method increases the bonding force between layers and can achieve the effect of different pore sizes between inner and outer layers, effectively reducing the risk of vascular stratification, and realizing the functions of preventing blood leakage and anticoagulation, and promoting tissue and endothelial regeneration.

[0071] The application further provides the artificial blood vessel prepared by the preparation method.

[0072] The application further provides application of the artificial blood vessel in the field of tissue engineering.

[0073] The artificial blood vessel is used for a blood vessel patch, a valved blood vessel and a heart valve.

[0074] The application realizes the integrated preparation of the artificial blood vessel with the outer layer aperture suitable for tissue growth and the inner layer aperture being dense by electrospinning of the matrix solution with two different concentrations and different flow rate parameters, so that the time cost and the economic cost are greatly saved. The preparation method of the artificial blood vessel provided by the application is simple, and the demand for the artificial blood vessel can be effectively met. After the artificial blood vessel is implanted into the body, the outer layer of the artificial blood vessel has the aperture suitable for tissue growth, which is conducive to cell adhesion and growth, and the dense inner layer aperture prevents blood leakage, so that the good performance meets the use of the artificial blood vessel.

[0075] In order to further understand the application, the artificial blood vessel with gradient inner and outer apertures and the preparation method and application thereof provided by the application are described below in combination with examples, and the protection scope of the application is not limited by the following examples.

[0076] Example 1

[0077] A chloroform-methanol solution with a volume ratio of 5:1 was placed in a glass vial, and polycaprolactone (PCL) particles were added in an amount of 0.1 g / mL and 0.25 g / mL, the bottle cap was tightened and sealed with a sealing film, and then the vial was placed on a magnetic stirrer for continuous stirring at room temperature and in the dark for 12 h. Electrospinning was performed on the above solution to prepare an electrospun PCL artificial blood vessel. The specific operation and parameters are as follows: the matrix solutions 1 and 2 were respectively filled into syringes 1 and 2, the syringes were inverted to discharge air bubbles, then 21G needle heads were installed and fixed, and a syringe pump was used for propulsion, the flow rate of the 0.1 g / mL PCL solution was set to 12 mL / h, and the flow rate of the 0.25 g / mL PCL solution was set to 720 mL / h 2to 0 mL / h, the flow rate of the 0.25 g / mL PCL solution was set to 0 mL / h with a uniform acceleration of 144 mL / h 2 to 12 mL / h, the high-voltage direct-current voltage was set to 10 kV, the receiving distance was set to 10 cm, the receiver was a grounded stainless steel rod with a diameter of 4 mm, the rotating speed of the stainless steel rod was 150 rpm, and the spinning time was 10 min.

[0078] Example 2

[0079] A chloroform-methanol solution with a volume ratio of 5:1 was placed in a glass vial, polycaprolactone (PCL) particles were added in an amount of 0.1 g / mL and 0.25 g / mL, the bottle cap was tightened and sealed with a sealing film, and then the vial was placed on a magnetic stirrer for continuous stirring at room temperature and in the dark for 12 h. Electrospinning was performed with the above solution to prepare an electrospun PCL artificial blood vessel. The specific operation and parameters are as follows: the matrix solutions 1 and 2 were respectively loaded into syringes 1 and 2, the syringes were inverted to discharge air bubbles, a 21G needle was then installed and fixed, a syringe pump was used for propulsion, the flow rate of the 0.1 g / mL PCL solution was set to 12 mL / h with a uniform deceleration of 360 mL / h 2 to 0 mL / h, the flow rate of the 0.25 g / mL PCL solution was set to 0 mL / h with a uniform acceleration of 144 mL / h 2 to 12 mL / h, the high-voltage direct-current voltage was set to 10 kV, the receiving distance was set to 10 cm, the receiver was a grounded stainless steel rod with a diameter of 4 mm, the rotating speed of the stainless steel rod was 150 rpm, and the spinning time was 10 min.

[0080] Example 3

[0081] A chloroform-methanol solution with a volume ratio of 5:1 was placed in a glass vial, polycaprolactone (PCL) particles were added in an amount of 0.1 g / mL and 0.25 g / mL, the bottle cap was tightened and sealed with a sealing film, and then the vial was placed on a magnetic stirrer for continuous stirring at room temperature and in the dark for 12 h. Electrospinning was performed with the above solution to prepare an electrospun PCL artificial blood vessel. The specific operation and parameters are as follows: the matrix solutions 1 and 2 were respectively loaded into syringes 1 and 2, the syringes were inverted to discharge air bubbles, a 21G needle was then installed and fixed, a syringe pump was used for propulsion, the flow rate of the 0.1 g / mL PCL solution was set to 12 mL / h with a uniform deceleration of 240 mL / h 2 to 0 mL / h, the flow rate of the 0.25 g / mL PCL solution was set to 0 mL / h with a uniform acceleration of 144 mL / h 2 to 12 mL / h, the high-voltage direct-current voltage was set to 10 kV, the receiving distance was set to 10 cm, the receiver was a grounded stainless steel rod with a diameter of 4 mm, the rotating speed of the stainless steel rod was 150 rpm, and the spinning time was 10 min.

[0082] Example 4

[0083] A chloroform-methanol solution with a volume ratio of 5:1 was placed in a glass vial, and polycaprolactone (PCL) particles were added in an amount of 0.1 g / mL and 0.25 g / mL. The vial was tightly capped and sealed with a sealing film, and then placed on a magnetic stirrer for continuous stirring at room temperature and in the dark for 12 h. Electrospinning was performed with the above solution to prepare an electrospun PCL artificial blood vessel. The specific operation and parameters were as follows: the substrate solutions 1 and 2 were respectively loaded into syringes 1 and 2, the syringes were inverted to discharge air bubbles, and then a 21G needle was installed and fixed. An injection pump was used for propulsion, the flow rate of the 0.1 g / mL PCL solution was set to 12 mL / h with a uniform deceleration of 144 mL / h 2 to 0 mL / h, and the flow rate of the 0.25 g / mL PCL solution was set to 0 mL / h with a uniform acceleration of 144 mL / h 2 to 12 mL / h, the high-voltage direct current voltage was set to 10 kV, the receiving distance was set to 10 cm, the receiver was a grounded stainless steel rod with a diameter of 4 mm, the rotating speed of the stainless steel rod was 150 rpm, and the spinning time was 10 min.

[0084] Example 5

[0085] A chloroform-methanol solution with a volume ratio of 5:1 was placed in a glass vial, and polycaprolactone (PCL) particles were added in an amount of 0.1 g / mL and 0.25 g / mL. The vial was tightly capped and sealed with a sealing film, and then placed on a magnetic stirrer for continuous stirring at room temperature and in the dark for 12 h. Electrospinning was performed with the above solution to prepare an electrospun PCL artificial blood vessel. The specific operation and parameters were as follows: the substrate solutions 1 and 2 were respectively loaded into syringes 1 and 2, the syringes were inverted to discharge air bubbles, and then a 21G needle was installed and fixed. An injection pump was used for propulsion, the flow rate of the 0.1 g / mL PCL solution was set to 12 mL / h with a uniform deceleration of 144 mL / h 2 to 0 mL / h, and the flow rate of the 0.25 g / mL PCL solution was set to 0 mL / h with a uniform acceleration of 144 mL / h 2 to 12 mL / h, the high-voltage direct current voltage was set to 10 kV, the receiving distance was set to 10 cm, the receiver was a grounded stainless steel rod with a diameter of 4 mm, the rotating speed of the stainless steel rod was 150 rpm, and the spinning time was 10 min.

[0086] The specific parameters of Examples 1-5 are shown in Table 1

[0087] Table 1: Summary of specific parameters of Examples 1-5

[0088]

[0089] Comparative Example 1

[0090] A chloroform-methanol solution with a volume ratio of 5:1 was placed in a glass vial, and polycaprolactone (PCL) particles were added in an amount of 0.1 g / mL. The vial was tightly capped with a sealing film, and then placed on a magnetic stirrer for continuous stirring at room temperature and in the dark for 12 h. Electrospinning was performed with the above solution to prepare an electrospun PCL artificial blood vessel. The specific operation and parameters were as follows: the substrate solutions 1 and 2 were respectively loaded into syringes 1 and 2, the syringes were inverted to remove air bubbles, and then a 21G needle was installed and fixed. An injection pump was used for propulsion, the flow rate of the 0.1 g / mL PCL solution in syringe 1 was set to 12 mL / h at a uniform deceleration of 240 mL / h 2 to 0 mL / h, and the flow rate of the 0.1 g / mL PCL solution in syringe 2 was set to 0 mL / h at a uniform acceleration of 144 mL / h 2 to 12 mL / h, the high-voltage direct current voltage was set to 10 kV, the receiving distance was set to 10 cm, the receiver was a grounded stainless steel rod with a diameter of 4 mm, the rotating speed of the stainless steel rod was 150 rpm, and the spinning time was 10 min.

[0091] Comparative Example 2

[0092] A chloroform-methanol solution with a volume ratio of 5:1 was placed in a glass vial, and polycaprolactone (PCL) particles were added in an amount of 0.25 g / mL. The vial was tightly capped with a sealing film, and then placed on a magnetic stirrer for continuous stirring at room temperature and in the dark for 12 h. Electrospinning was performed with the above solution to prepare an electrospun PCL artificial blood vessel. The specific operation and parameters were as follows: the substrate solutions 1 and 2 were respectively loaded into syringes 1 and 2, the syringes were inverted to remove air bubbles, and then a 21G needle was installed and fixed. An injection pump was used for propulsion, the flow rate of the 0.25 g / mL PCL solution in syringe 1 was set to 12 mL / h at a uniform deceleration of 240 mL / h 2 to 0 mL / h, and the flow rate of the 0.25 g / mL PCL solution in syringe 2 was set to 0 mL / h at a uniform acceleration of 144 mL / h 2 to 12 mL / h, the high-voltage direct current voltage was set to 10 kV, the receiving distance was set to 10 cm, the receiver was a grounded stainless steel rod with a diameter of 4 mm, the rotating speed of the stainless steel rod was 150 rpm, and the spinning time was 10 min.

[0093] Comparative Example 3

[0094] A chloroform-methanol solution with a volume ratio of 5:1 was placed in a glass vial, polycaprolactone (PCL) particles were configured into matrix solution 1 and matrix solution 2 at concentrations of 0.1 g / mL and 0.25 g / mL, the bottle cap was tightened and sealed with a sealing film, and then the vial was placed on a magnetic stirrer at room temperature and continuously stirred in the dark for 12 h. Electrospinning was performed with the above solution to prepare an electrospun PCL artificial blood vessel. The specific operation and parameters are as follows: matrix solutions 1 and 2 were respectively loaded into syringes 1 and 2, the syringes were inverted to discharge the bubbles, and then a 21G needle was installed and fixed, syringe 1 was first opened to push forward, the flow rate of syringe 1 was set to 12 mL / h, and the speed was uniformly reduced to 240 mL / h 2 0 mL / h, and when the spinning of solution 1 was completed, the injection pump of syringe 2 was opened, the flow rate of syringe 2 was set to 0 mL / h, and the speed was uniformly increased to 144 mL / h 2 12 mL / h, the high-voltage direct current voltage was set to 10 kV, the receiving distance was set to 10 cm, the receiver was a grounded stainless steel rod with a diameter of 4 mm, the rotating speed of the stainless steel rod was 150 rpm, and the spinning time was 20 min.

[0095] Table 2

[0096]

[0097] The tensile strength and suture strength of the artificial blood vessels prepared in Examples 1-5 and Comparative Examples 1-3 were characterized and detected, and the specific method was as follows:

[0098] 1. Mechanical characterization

[0099] The axial tensile strength and radial tensile strength of the artificial blood vessels prepared in Examples 1-5 and Comparative Examples 1-3 were tested by an Instron-3345 tensile machine.

[0100] The axial mechanical test method was as follows: the upper end and the lower end of the blood vessel with a length of 2.5 cm were fixed to the upper and lower clamps of the tensile machine, the distance between the upper and lower clamps was controlled to be 1 cm, then the parameters of the sample were input, the stretching speed was adjusted to 20 mm / min, the tensile force of the software was adjusted to zero, the tensile machine was started until the sample was completely broken, and the Young's modulus, stress and maximum breaking elongation of the sample were obtained according to the stress-strain curve.

[0101] The radial mechanical test method was as follows: the sample with a length of about 5 mm was fixed to the tensile machine through a steel ring, the parameters of the sample were input, the stretching speed was adjusted to 10 mm / min, the tensile force of the software was adjusted to zero, the tensile machine was started until the sample was completely broken, and the Young's modulus, stress and maximum breaking elongation of the sample were obtained according to the stress-strain curve.

[0102] The test method for delamination force is as follows: take a 50 mm long artificial blood vessel, cut it longitudinally into a flat shape, and physically separate the outer layer and the support from the bottom layer to form two approximately 15 mm long tabs. Each tab is clamped with a clamp, and the upper layer tab is pulled at a speed of 20 mm / min, while the force is measured and the peak force of the sample is recorded to evaluate the bonding strength between the outer layer and the inner layer.

[0103] 2. Suture strength characterization

[0104] The flat end (0°) suture strength of the artificial blood vessels prepared in Examples 1-5 and Comparative Examples 1-3 was tested according to the ISO 7198:2016 A.5.7 suture strength detection standard, and the test method was as follows: the blood vessel was cut into small pieces with a length of about 1 cm, and a 6-0 medical suture was passed through the sample tube wall about 2 mm away from the horizontal end of the blood vessel, the suture was knotted to form a closed loop, the knotted suture was clamped with the air pump clamp of the tensile testing machine, and the other end of the blood vessel was also clamped with the air pump clamp of the tensile testing machine, the tensile testing machine was balanced and zeroed; the stretching speed was 8.00 mm / min, and the stretching was carried out until the 6-0 suture penetrated the sample end.

[0105] 3. Anticoagulant property detection

[0106] Platelet adhesion and activation evaluation: the membranes of Examples 1-5 and Comparative Examples 1-3 were cut into the size of a 48-well plate, 500 μL of platelet-rich plasma (PRP) was added to each well, and incubated at 37°C for 2 hours, then the adhered platelets and activated platelets were quantified using lactate dehydrogenase (LDH) kit and soluble P-selectin ELISA kit respectively. The absorbance value of the well plate at 490 nm was detected by a multifunctional enzyme label instrument to represent the data of platelet adhesion, and the larger the data, the more platelets adhered; the absorbance value of the well plate at 450 nm was detected by a multifunctional enzyme label instrument to represent the data of platelet activation, and the larger the data, the more activated platelets.

[0107] 4. Data analysis

[0108] The data was analyzed using GraphPad Prism 9.0.0 software, and all data was expressed as mean ± standard error.

[0109] Table 3. Mechanical characterization of blood vessels (parallel experiments, n = 3, mean ± standard error)

[0110]

[0111] The results of Table 3 show that, compared with Comparative Example 3, Examples 1-5 have certain significant changes in the radial maximum stress, radial Young's modulus, axial maximum stress, axial Young's modulus, suturing strength, and burst pressure; the delamination force test results show that Example 3 is significantly better than Comparative Example, especially significantly better than Comparative Example 3, indicating that the mechanical properties of the integrated tube are better than those of the simple inner-outer layer superimposed textile artificial blood vessel and are not prone to delamination.

[0112] Figure 2 The light absorption value of the detection hole plate of the multifunctional enzyme label instrument at 490 nm represents the adhesion data of the platelets; Figure 3 The light absorption value of the detection hole plate of the multifunctional enzyme label instrument at 450 nm represents the activation data of the platelets.

[0113] The results of Figure 2 and Figure 3 show that Comparative Example 2 has a significant difference in platelet adhesion from the other groups, indicating that the inner layer of coarse filaments is not conducive to anti-platelet adhesion. The platelet activation data show that Examples 3-5 and Comparative Examples 1 and 3 all have platelet activation, and there is no significant difference, while Comparative Example 2 has a significant difference from the other groups. The above results show that the examples can adjust the speed of the matrix solution to make the blood vessel have good mechanical properties while also reducing the adhesion and activation of platelets in the inner layer of electrospun filaments, and increase the anticoagulant properties of the blood vessel.

[0114] The above only describes the preferred embodiments of the present application, and it should be noted that, for ordinary skilled persons in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A method for preparing an artificial blood vessel with gradient inner and outer pore sizes, characterized in that, Includes the following steps: A) Prepare first matrix solutions and second matrix solutions with different concentrations respectively; The first matrix solution and the second matrix solution comprise polymeric materials; The concentration of the first matrix solution is less than the concentration of the second matrix solution; B) The first matrix solution and the second matrix solution are mixed after being flowed in their respective modes to obtain a mixed matrix solution; The flow pattern of the first matrix solution is as follows: it operates with a first initial flow rate and gradually reduces the flow rate by deceleration; The flow pattern of the second matrix solution is as follows: the flow rate is gradually increased by combining a second initial flow rate with acceleration; The first initial flow velocity is higher than the second initial flow velocity; C) The mixed matrix solution is electrospun on the surface of the receiver to obtain an integrated artificial blood vessel.

2. The preparation method according to claim 1, characterized in that, The polymeric material includes at least one or a mixture of several of the following in any proportion: polycaprolactone, polylactic acid, polyurethane, polylactic acid-glycolic acid copolymer, poly-L-lactide-caprolactone, and poly(p-dioxane-hexanone).

3. The preparation method according to claim 1, characterized in that, The solvents used to prepare the first matrix solution and the second matrix solution are selected from at least one or a mixture of several of hexafluoroisopropanol, chloroform, and methanol in any proportion.

4. The preparation method according to claim 1, characterized in that, The concentration of the first matrix solution is 0.1~0.3 g / mL; The concentration of the second matrix solution is 0.2~0.5 g / mL.

5. The preparation method according to claim 1, characterized in that, The initial flow rate is 10~12 mL / h, and the deceleration is -144~-720 mL / h. 2 ; The second initial flow rate is 0~2 mL / h, and the acceleration is 144~720 mL / h. 2 .

6. The preparation method according to claim 1, characterized in that, The electrospinning process uses a high-voltage DC voltage of 10~12kV, a distance of 10~15cm from the receiver, and a spinning time of 10~12min.

7. The preparation method according to claim 1, characterized in that, The receiver is a grounded stainless steel rod with a diameter of 2-8 mm; The receiver rotates at a speed of 120~180 rpm.

8. An artificial blood vessel with gradient inner and outer pore sizes prepared by the preparation method according to any one of claims 1 to 7, characterized in that, The artificial blood vessel has an integrated structure.

9. The application of the artificial blood vessel as described in claim 8 in the preparation of tissue engineering materials.

10. The application according to claim 9, characterized in that, The artificial blood vessels are used for vascular patches, valved blood vessels, and heart valves.

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