CN115607744A

Movatterモバイル変換

Info

Publication number: CN115607744A
Application number: CN202211523376.1A
Authority: CN
Inventors: 张海军
Original assignee: Shandong Rientech Medical Technology Co ltd
Current assignee: Shandong Rientech Medical Technology Co ltd
Priority date: 2022-12-01
Filing date: 2022-12-01
Publication date: 2023-01-17
Anticipated expiration: 2042-12-01
Also published as: CN115607744B

Abstract

The invention relates to the field of medical implant materials, in particular to a segmented functionalized artificial blood vessel. In order to solve the existing problems of thrombus and intimal hyperplasia in artificial blood vessel transplantation and promote blood vessel repair, the invention prepares a block functionalized artificial blood vessel, the artificial blood vessel is divided into three sections according to different functions, an anticoagulation and anti-hyperplasia section, an anticoagulation and endothelialization promoting section and an anticoagulation and anti-hyperplasia section, the function distribution caters to the occurrence part of postoperative complications, the thrombus and the tissue hyperplasia are reduced, and meanwhile, the middle section anticoagulation can promote cell endothelialization and accelerate the tissue repair.

Description

Block functionalized artificial blood vessel

Technical Field

The invention relates to the field of medical implant materials, in particular to a segmented functionalized artificial blood vessel.

Background

Cardiovascular diseases (CVDs) have become an important cause of death in the world. According to the world health organization data, about 1790 million die each year from cardiovascular disease. Vascular replacement or revascularization is the most common surgical procedure, and with the increasing aging population and the concomitant chronic diseases such as uremia, diabetes and the like, the clinical need for vascular grafts with long-term patency is increasing.

Engineering of single-cell-layered vascular and tissue-engineered vascular implants composed of natural and synthetic polymeric materials has been a major advance over decades, but many challenges remain in terms of antithrombotic formation, rapid endothelialization, modulation of inflammatory responses, and inhibition of intimal hyperplasia and calcification.

Techniques for manufacturing artificial blood vessels generally include electrospinning, decellularization, lyophilization, and 3D printing and bioprinting, wherein electrospinning is widely used due to its simplicity and versatility. Various natural, synthetic or hybrid materials can be electrospun into tubular structures for revascularization. In addition, by adjusting the polymer concentration, voltage, flow rate and other parameters of electrostatic spinning, the vascular graft with controllable fiber diameter, porosity, inner diameter, mechanical property and components can be easily obtained. By modifying the electrostatic spinning material, biological behaviors such as cell adhesion, infiltration and the like can be obviously improved, and tissue regeneration is promoted.

Aiming at the problems that the artificial blood vessel transplantation suture part is easy to generate thrombus and tissue hyperplasia, the middle section is easy to generate thrombus and the requirement for promoting the growth of autologous blood vessels, the invention prepares the artificial blood vessel with the functions of block anticoagulation and anti-hyperplasia, anticoagulation and promotion of endothelialization and anticoagulation through the electrostatic spinning technology.

Disclosure of Invention

Aiming at the problems that the artificial blood vessel transplantation suture part is easy to generate thrombus and tissue hyperplasia, the middle section is easy to generate thrombus and the requirement for promoting the growth of autologous blood vessels, the invention prepares the artificial blood vessel with the functionalized blocks, the thrombus and the tissue hyperplasia are reduced in a targeted way, and meanwhile, the middle section anticoagulation can promote the endothelialization of cells and accelerate the self-repair of the blood vessel.

In order to realize the purpose, the invention provides the following technical scheme:

a segmented functionalized artificial blood vessel can be divided into three segments according to functions: S1-S2-S3 respectively correspond to the functions of anticoagulation and antiproliferation, anticoagulation and endothelialization, anticoagulation and antiproliferation, and the preparation method comprises the following steps:

dissolving poly L-lactide-caprolactone in hexafluoroisopropanol organic solvent to prepare PLCL spinning solution;

preparing a heparin aqueous solution and an ethanol solution of rapamycin in the step (2), sequentially mixing the heparin aqueous solution and the ethanol solution of rapamycin in the step (1), adding a cross-linking agent to prepare an anticoagulant and antiproliferative functional solution L_S1 ;

After the heparin aqueous solution is prepared in the step (3), the heparin aqueous solution, CD33, CD34 and cell growth factor VEGF are added into the PLCL spinning solution and are blended with the cross-linking agent to prepare the anticoagulant and endothelialization promoting functional solution L_S2 ；

Step (4) of preparing the functional solution L of the anticoagulant and antiproliferative segment_S3 The preparation method is the same as L_S1 ；

Step (5) according to L_S1 ，L_S2 ，L_S3 The artificial blood vessel with the functions of promoting endothelialization, anticoagulation and anti-hyperplasia is prepared by electrostatic spinning in the using sequence.

Further, the concentration of the PLCL spinning solution in the step (1) is 0.1-1.0g/mL;

further, L in the step (2)_S1 The concentration of the heparin is 50-200mg/mL;

further, L in the step (2)_S1 The concentration of the rapamycin solution is 30-50 mu g/mL;

further, the cross-linking agent in the step (3) is one or more of 1, 3-dimethylaminopropyl-3-ethylcarbodiimide, N-hydroxysuccinimide, 2-morpholine ethanesulfonic acid, carbodiimide, genipin and polyethylene glycol glycerol ether, and the mass concentration of the cross-linking agent is 1.0-5.0 wt%;

further, step (3) said L_S2 The concentration of the heparin is 50-200mg/mL;

further, said L of step (3)_S2 The mass concentrations of the medium CD33 and the CD34 are respectively 0.2-2.0 mug/mL;

in a further aspect of the present invention,l in the step (3)_S2 The concentration of the medium cell growth factor VEGF solution is 10-50 mug/mL;

further, the electrostatic spinning parameters in the step (5) are set as follows: the flow rate is 0.1-1.0mL/h, the voltage is 15-20kV, the receiving distance is 10-20cm, and the three-section collection time is respectively as follows: 0.5-1.0h,1.5-3.0h and 0.5-1.0h;

further, the diameter of the artificial blood vessel in the step (5) is 0.5-2.0mm, and the wall thickness is 100-150 μm;

further, the length of the S1 section and the S3 section of the artificial blood vessel is 0.1-0.5mm, and the length of the S2 section can be determined according to the length of a lesion.

The invention has the beneficial effects that:

the invention provides a preparation method of a block functionalized artificial blood vessel aiming at the problems that thrombus and intimal hyperplasia are more likely to occur at two ends after an artificial blood vessel transplantation operation and anticoagulation is needed in the middle section and the blood vessel repair is accelerated, wherein the artificial blood vessel is divided into an anticoagulation antiproliferation-anticoagulation endothelialization-anticoagulation antiproliferation section, and the target reduction of the complications and the promotion of blood vessel endothelialization and self-repair are simultaneously realized.

Drawings

FIG. 1 is a schematic representation of the present invention, wherein 1 represents the S1 segment, 2 represents the S2 segment, and 3 represents the S3 segment;

FIG. 2 is an absorbance value test result;

FIG. 3 is an endothelial cell culture curve;

figure 4 is a histogram of intimal hyperplasia thickness.

Detailed Description

Example 1

(1) Dissolving PLCL in hexafluoroisopropanol organic solvent to prepare 0.1g/mL PLCL spinning solution; dissolving heparin in water, dissolving rapamycin in a little ethanol, then respectively adding the dissolved rapamycin into the spinning solution, wherein the concentrations of the heparin and the rapamycin in the spinning solution are respectively 200mg/mL and 50 mu g/mL, then adding 2wt% of cross-linking agent genipin, and uniformly mixing to obtain a spinning solution L_S1 。

(2) Dissolving PLCL in hexafluoroisopropanol organic solvent to prepare 0.1g/mL PLCL spinning solution; however, the device is not suitable for use in a kitchenThen dissolving heparin in water, adding the PLCL solution, mixing uniformly, and adding CD33, CD34 and cell growth factor VEGF respectively to make the concentrations of CD33 and CD34 respectively be 1.0 mug/mL, the concentration of VEGF be 20 mug/mL and the concentration of heparin be 200mg/mL. Adding 2wt% of cross-linking agent genipin, and mixing to obtain spinning solution L_S2 。

(3) Spinning solution L_S3 And L_S1 The preparation method is the same.

(4) The electrostatic spinning parameters are set as follows: flow rate 0.5mL/h, voltage 18kV, receiving distance 15cm, according to L_S1 ，L_S2 ，L_S3 The using sequence of (1) is electrostatic spinning, and the three-stage collection time is respectively as follows: 1.0h,2h and 1.0h, and the artificial blood vessel with the functions of promoting endothelialization, anticoagulation and anti-hyperplasia is prepared.

Example 2

(1) Dissolving PLCL in hexafluoroisopropanol organic solvent to prepare 0.1g/mL PLCL spinning solution; dissolving heparin in water, dissolving rapamycin in a little ethanol, then respectively adding the dissolved rapamycin into the spinning solution, wherein the concentrations of the heparin and the rapamycin in the spinning solution are respectively 50mg/mL and 30 mu g/mL, then adding 1wt% of crosslinking agent genipin, and uniformly mixing to obtain a spinning solution L_S1 。

(2) Dissolving PLCL in hexafluoroisopropanol organic solvent to prepare 0.1g/mL PLCL spinning solution; then dissolving heparin in water, adding the PLCL solution, mixing uniformly, and adding CD33, CD34 and cell growth factor VEGF respectively to make the concentrations of CD33 and CD34 respectively 0.2 mu g/mL, VEGF 10 mu g/mL and heparin 50mg/mL. Adding 1wt% of crosslinking agent genipin, and mixing uniformly to obtain spinning solution L_S2 。

(3) Spinning solution L_S3 And L_S1 The preparation method is the same.

(4) Setting electrostatic spinning parameters as follows: the flow rate was 1.0mL/h, the voltage was 15kV, the receiving distance was 20cm, in terms of L_S1 ，L_S2 ，L_S3 The using sequence of (1) is electrostatic spinning, and the three-stage collection time is respectively as follows: 1.0h,2h and 1.0h. The artificial blood vessel with the functions of promoting endothelialization, anticoagulation and anti-hyperplasia can be prepared.

Example 3

(1) Dissolving PLCL in hexafluoroisopropanol organic solvent to prepare 0.8g/mL PLCL spinning solution; dissolving heparin in water, dissolving rapamycin in a little ethanol, then respectively adding the dissolved rapamycin into the spinning solution, wherein the concentrations of the heparin and the rapamycin in the spinning solution are respectively 180mg/mL and 40 mu g/mL, then adding 1wt% of crosslinking agent genipin, and uniformly mixing to obtain a spinning solution L_S1 。

(2) Dissolving PLCL in hexafluoroisopropanol organic solvent to prepare 1.0g/mL PLCL spinning solution; then dissolving heparin in water, adding the PLCL solution, mixing uniformly, and adding CD33, CD34 and cell growth factor VEGF respectively to make the concentrations of CD33 and CD34 respectively 1.8 mug/mL,VEGF 40 mug/mL and heparin 180mg/mL. Adding 1wt% of crosslinking agent genipin, and mixing uniformly to obtain spinning solution L_S2 。

(3) Spinning solution L_S3 And L_S1 The preparation method is the same.

(4) The electrostatic spinning parameters are set as follows: flow rate 0.1mL/h, voltage 20kV, receiving distance 10cm, according to L_S1 ，L_S2 ，L_S3 The using sequence of (1) is electrostatic spinning, and the three-stage collecting time is respectively as follows: 1.0h,2h and 1.0h. The artificial blood vessel with the functions of promoting endothelialization, anticoagulation and anti-hyperplasia is prepared.

Example 4

The artificial blood vessels prepared in examples and comparative examples were subjected to a whole blood clotting time test, and the absorbance of the supernatant after 10min, 30min and 60min after the addition of calcium chloride to the whole blood was measured. The larger the absorbance, the larger the absorbance of red blood in the supernatant, and the larger the hemoglobin content in the supernatant, the fewer the blood cells that produce blood coagulation, and the slower the coagulation rate. Therefore, the greater the absorbance value of the supernatant of the sample to be detected, the greater the coagulation index and the better the anticoagulation performance of the material. The results of the absorbance value test are shown in FIG. 2.

Example 5

Take 5.0X 10⁶ Endothelial cells of high density were inoculated onto the artificial blood vessels prepared in examples 1-3 and comparative examples 1-5 (examples 1-3 correspond to experimental groups 1-3, respectively, and comparative examples 1-5 correspond to experimental groups 4-8, respectively, and fetal bovine at 1640+20%The serum is used as culture solution for culturing. The number of cells was counted by microscopic observation and microtitre analysis to prepare a growth curve, and the results are shown in FIG. 3.

Example 6

And (3) obtaining transplanted blood vessels after conventional breeding for 4 weeks, observing intimal hyperplasia conditions of the transplanted blood vessels by HE (human immunodeficiency Virus) staining, and measuring the intimal thickness of the transplanted blood vessels by a computer image analysis system. The results of the experiment are shown in FIG. 4.

Comparative example 1

(1) Dissolving PLCL in hexafluoroisopropanol organic solvent to prepare 0.05g/mL PLCL spinning solution; dissolving heparin in water, dissolving rapamycin in a little ethanol, then respectively adding the dissolved rapamycin into the spinning solution, wherein the concentrations of the heparin and the rapamycin in the spinning solution are respectively 5mg/mL and 5 mu g/mL, then adding 1wt% of crosslinking agent genipin, and uniformly mixing to obtain a spinning solution L_S1 。

(2) Dissolving PLCL in hexafluoroisopropanol organic solvent to prepare 0.05g/mL PLCL spinning solution; then, heparin was dissolved in water, and the above PLCL solution was added, and after mixing uniformly, CD33, CD34 and VEGF, which were cell growth factors, were added so that the concentrations of CD33 and CD34 were 0.1. Mu.g/mL, respectively, the concentration of VEGF was 5. Mu.g/mL, and the concentration of heparin was 5mg/mL. Adding 1wt% of crosslinking agent genipin, and mixing uniformly to obtain spinning solution L_S2 。

(3) Spinning solution L_S3 And L_S1 The preparation method is the same.

(4) The electrostatic spinning parameters are set as follows: flow rate 0.5mL/h, voltage 18kV, receiving distance 15cm, according to L_S1 ，L_S2 ，L_S3 The using sequence of (1) is electrostatic spinning, and the three-stage collecting time is respectively as follows: 1.0h,2h and 1.0h. The artificial blood vessel with the functions of promoting endothelialization, anticoagulation and anti-hyperplasia is prepared.

Comparative example 2

(1) Dissolving PLCL in hexafluoroisopropanol organic solvent to prepare 0.1g/mL PLCL spinning solution; then adding 2wt% of crosslinking agent genipin, and uniformly mixing to obtain a spinning solution.

(2) The electrostatic spinning parameters are set as follows: carrying out electrostatic spinning at the flow rate of 0.5mL/h, the voltage of 18kV and the receiving distance of 15cm for 2h to prepare a blank artificial blood vessel.

Comparative example 3

(1) Dissolving PLCL in hexafluoroisopropanol organic solvent to prepare 0.1g/mL PLCL spinning solution; dissolving heparin in water, dissolving rapamycin in a little ethanol, then respectively adding the dissolved rapamycin into the spinning solution, wherein the concentrations of the heparin and the rapamycin in the spinning solution are respectively 200mg/mL and 50 mu g/mL, then adding 2wt% of crosslinking agent genipin, and uniformly mixing to obtain a spinning solution L_S1 。

(2) The electrostatic spinning parameters are set as follows: carrying out electrostatic spinning at the flow rate of 0.5mL/h, the voltage of 18kV and the receiving distance of 15cm, and collecting for 4h to obtain the artificial blood vessel.

Comparative example 4

(1) Dissolving PLCL in hexafluoroisopropanol organic solvent to prepare 0.1g/mL PLCL spinning solution; then dissolving heparin in water, adding the PLCL solution, mixing uniformly, and adding CD33, CD34 and cell growth factor VEGF respectively to make the concentrations of CD33 and CD34 respectively 1.0 mug/mL,VEGF concentration 20 mug/mL and heparin concentration 200mg/mL. Adding 2wt% of crosslinking agent genipin, and mixing uniformly to obtain spinning solution L_S2 。

(2) The electrostatic spinning parameters are set as follows: the flow rate is 0.5mL/h, the voltage is 18kV, the receiving distance is 15cm, the collecting time is 4h, and the artificial blood vessel is prepared.

Comparative example 5

(1) Dissolving PLCL in hexafluoroisopropanol organic solvent to prepare 0.1g/mL PLCL spinning solution; then dissolving heparin in water, dissolving rapamycin in a little ethanol, mixing uniformly, and then adding CD33, CD34 and cell growth factor VEGF respectively, wherein the concentrations of heparin and rapamycin in the spinning solution are respectively 200mg/mL and 50 μ g/mL, the concentrations of CD33 and CD34 are respectively 1.0 μ g/mL, the concentration of VEGF is 20 μ g/mL, and the concentration of heparin is 200mg/mL. Adding 2wt% of cross-linking agent genipin, and mixing uniformly to obtain a spinning solution.

(2) The electrostatic spinning parameters are set as follows: the flow rate was 0.5mL/h, the voltage was 18kV, the reception distance was 15cm, and the collection time was 4h. The artificial blood vessel with the functions of promoting endothelialization, anticoagulation and anti-hyperplasia is prepared.

And (4) analyzing results: as can be seen from the results of the absorbance value test in FIG. 2, the anticoagulation effect of the

experimental groups

1,2,3,6,7,8 is better, which shows that the anticoagulation effect of the artificial blood vessels prepared in examples 1-3 and comparative examples 3-5 is better; as can be seen from the endothelial cell culture curve in FIG. 3, the endothelial cell growth curves of examples 1-3 and comparative example 4 are better in number, which proves that the artificial blood vessel of the vegetation of the present invention can promote the growth of endothelial cells; as can be seen from the histogram of the intimal hyperplasia thickness in FIG. 4, the intimal hyperplasia inhibition effects obtained in the

experimental groups

1,2,3,6 and 8 are relatively good, and the effects obtained in the

experiments

4,5 and 7 are relatively poor, which proves that the artificial blood vessels prepared in examples 1-3 and comparative examples 3 and 8 have the intimal hyperplasia inhibition effects. Therefore, the artificial blood vessels prepared in theembodiments 1 to 3 have better anticoagulation, anti-hyperplasia and endothelialization promoting effects in combination with experimental results, and prove that the block functionalized artificial blood vessel prepared by the invention can effectively reduce complications in a targeted manner, and promote endothelialization and self-repair of blood vessels.

Claims

1. A segmented functionalized artificial blood vessel is characterized in that the artificial blood vessel is divided into three segments according to different functions: S1-S2-S3 respectively correspond to the functions of anticoagulation and antiproliferation, anticoagulation and endothelialization, anticoagulation and antiproliferation, and the preparation method comprises the following steps:

step (1): dissolving poly L-lactide-caprolactone in hexafluoroisopropanol organic solvent to prepare PLCL spinning solution;

step (2): respectively preparing heparin aqueous solution and rapamycin ethanol solution, sequentially adding the heparin aqueous solution and the rapamycin ethanol solution into the PLCL spinning solution prepared by the method in the step (1), uniformly mixing, and then adding a cross-linking agent to prepare the anticoagulant and antiproliferative functional solution L_S1 ；

And (3): after preparing the heparin aqueous solution, adding the heparin aqueous solution, CD33, CD34 and VEGF (cell growth factor) into the PLCL spinning solution prepared by the method in the step (1), uniformly mixing, adding a cross-linking agent, and preparing an anticoagulant and endothelialization promoting functional solution L_S2 ；

And (4): preparing functional solution L of anticoagulant and antiproliferative segment_S3 The method is as L_S1 A preparation method;

and (5): according to L_S1 ，L_S2 ，L_S3 The diameter of the artificial blood vessel is 0.5-2.0mm, the wall thickness is 100-150 mu m, and the lengths of the S1 section and the S3 section are 0.1-0.5mm respectively.

2. The block functionalized artificial blood vessel of claim 1, wherein the concentration of the PLCL spinning solution in the step (1) is 0.1-1.0g/mL.

3. The block functionalized artificial blood vessel of claim 1, wherein L in step (2)_S1 The heparin concentration of the heparin solution is 50-200mg/mL.

4. The block functionalized artificial blood vessel of claim 1, wherein L in step (2)_S1 The rapamycin concentration in the rapamycin ethanol solution is 30-50 mug/mL.

5. The block functionalized artificial blood vessel according to claim 1, wherein the crosslinking agent is one or more of 1, 3-dimethylaminopropyl-3-ethylcarbodiimide, N-hydroxysuccinimide, 2-morpholine ethanesulfonic acid, carbodiimide, genipin and polyethylene glycol glyceryl ether, and the mass concentration of the crosslinking agent is 1.0wt% to 5.0wt%.

6. The block functionalized vascular prosthesis of claim 1, wherein step (3) provides L_S2 The heparin concentration of the heparin aqueous solution is 50-200mg/mL.

7. The block functionalized vascular prosthesis of claim 1, wherein step (3) provides L_S2 The mass concentration of the medium CD33 and the mass concentration of the CD34 are respectively 0.2-2.0 mu g/mL.

8. The block functionalized artificial blood vessel of claim 1, wherein L in step (3)_S2 The concentration of the VEGF solution of the medium cell growth factor is 10-50 mu g/mL.

9. The block functionalized artificial blood vessel according to claim 1, wherein the electrospinning parameters of step (5) are set as follows: the flow rate is 0.1-1.0mL/h, the voltage is 15-20kV, the receiving distance is 10-20cm, and the three-stage collection time is respectively as follows: 0.5-1.0h,1.5-3.0h and 0.5-1.0h.