CN114642518A - Biocompatible vascular patch - Google Patents

Abstract

The present invention provides a biocompatible vascular patch comprising: the composite material comprises a middle layer, an inner layer and an outer layer, wherein the inner layer and the outer layer are attached to two opposite sides of the middle layer respectively, the inner layer is provided with a porous structure formed by stacking continuous nano fibers made of a first elastic polymer material, and the porosity is 60% -80%; the outer layer has a porous structure formed by a stack of truncated nanofibers having a porosity of 80% to 90%, and the nanofibers are made of a second elastic polymer material; the intermediate layer is a water-impermeable elastic layer formed from a third elastic polymeric material. The biocompatible vascular patch of the invention can achieve the effects of reducing thrombosis, instantly stopping bleeding and being easily matched with different repair parts.

Description

Biocompatible Vascular Patches

technical field

The present invention relates to the field of biomedicine, in particular to a biocompatible vascular patch.

Background technique

The carotid arteries are the main arteries on either side of the neck that supply blood to the brain. Carotid occlusive disease is caused by atherosclerosis. Atherosclerotic plaque builds up on the walls of arteries, narrowing them (stenosis) or making the walls so thick that they completely block blood flow (occlusion). The formation and accumulation of atherosclerotic plaque increases the risk of stroke, and about 20 percent of strokes are associated with atherosclerosis.

Clinical treatment is usually performed by carotid endarterectomy (CEA) combined with plaque angioplasty. This procedure, which removes the plaque that narrows the artery, is done through a small incision on the affected side of the neck; it involves opening the artery and then removing the plaque. After the plaque is removed, the artery is closed by suturing a vein or artificial material patch over the artery. The use of mesh closure reduces the risk of restenosis or occlusion. Expanded polytetrafluoroethylene (ePTFE) patch, polyester patch and autologous vein patch are the most commonly used patches in clinical stage.

Studies have shown that synthetic meshes have several advantages over intravenous meshes: reduced operative time, lower incidence of aneurysm expansion or mesh rupture, ease of use, and avoidance of site-of-use complications. However, few studies have shown whether the use of cardiovascular patches can shorten the time to hemostasis. On this subject, W.L. Gore Associates has introduced a new modified polytetrafluoroethylene patch (ACUSEAL) over the past few years, which the company claims has Use can greatly shorten the hemostasis time. The patch has a three-layer structure, the middle layer of the three-layer structure is fluoropolymer, and the inner and outer layers are all ePTFE materials. The elasticity of this patch is insufficient to match the blood vessel to be repaired well, and the ePTFE layer in contact with blood will leave a local uneven surface after puncture, which is prone to thrombosis. Therefore, an artificial blood vessel patch that can reduce thrombus formation, has good elasticity, and can instantaneously stop bleeding at the position of the needle hole after the suture needle passes through has very important clinical significance.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a biocompatible vascular patch, comprising: an intermediate layer and an inner layer and an outer layer respectively attached to opposite sides of the intermediate layer, wherein:

the inner layer has a porous structure formed by stacking continuous nanofibers made of a first elastic polymer material, with a porosity of 60% to 80%;

the outer layer has a porous structure formed by stacks of truncated nanofibers with a porosity of 80% to 90%, and the nanofibers are made of a second elastic polymer material;

The intermediate layer is a water impermeable elastic layer formed of a third elastic polymer material.

In some embodiments of the first aspect of the present invention, the nanofibers of the inner layer have a diameter of 100 nm to 800 nm, the nanofibers of the outer layer have a diameter of 100 nm to 800 nm, and the truncated nanofibers in the outer layer have a diameter of 100 nm to 800 nm. The length of the fibers is 60-300 μm, for example, the average length of the truncated nanofibers can be 150 μm.

In some embodiments of the first aspect of the present invention, the first elastic polymer material, the second elastic polymer material, and the third elastic polymer material are each independently selected from: polylactic acid/polyhexamethylene Lactone copolymer, polycaprolactone, polyurethane, polylactic acid, polylactic acid glycolic acid copolymer, poly(2-oxazoline), silicone, polyamide-containing polymers, polyether urethane. In an exemplary embodiment of the first aspect of the present invention, the first elastic polymer material is polylactic acid/polycaprolactone copolymer, the second elastic polymer material is polyurethane, and the first elastic polymer material is polyurethane. The trielastic polymer material is polycaprolactone.

In some embodiments of the first aspect of the invention, the inner layer comprises an anticoagulant material, such as heparin, and, in some embodiments, the anticoagulant material is coated on the outer surface of the inner layer .

In some embodiments of the present invention, the inner layer is formed by electrospinning techniques. The outer layer is made by the following steps: (1) making the second elastic polymer material into a nanofiber membrane by electrospinning; (2) cutting the nanofiber membrane prepared in step (1) Cut and disperse in a solvent for homogenization treatment to obtain a nanofiber dispersion; (3) freeze-dry the nanofiber dispersion in a mold of a predetermined size to obtain a loose porous structure formed by stacking truncated nanofibers outer layer of the structure. The intermediate layer is made by a casting molding process, a fluid spraying process, a dip coating process, a cladding coating process or a vapor deposition process. The thickness of the inner layer is 0.1 to 3 mm, the thickness of the intermediate layer is 0.01 to 2 mm, and the thickness of the outer layer is 0.05 to 3 mm.

In another aspect, the present invention provides a method for preparing the biocompatible vascular patch of the first aspect of the present invention, comprising the steps of:

(i) separately providing the inner layer, the middle layer and the outer layer and placing the inner layer and the outer layer on opposite sides of the middle layer;

(ii) subjecting the inner layer, outer layer and intermediate layer placed in step (i) to thermal compression bonding, so that the inner layer and outer layer are attached to opposite sides of the intermediate layer; wherein, the The thermocompression bonding process is carried out at a temperature of 80-85° C. and a pressure of 10-12 kPa.

As used herein, the term "water impermeable" means that no fluid leakage occurs under the application of pressure greater than that of blood in the human body against the walls of a blood vessel.

As used herein, the term "inner layer" refers to the layer of the vascular patch of the present invention that contacts the bloodstream during use.

As used herein, the term "outer layer" refers to the layer of the vascular patch of the present invention which, in use, contacts the tissue at the site to be repaired.

The term "intermediate layer" as used herein refers to a layer sandwiched between an inner layer and an outer layer.

The term "continuous nanofibers" as used herein refers to nanofibers formed directly by electrospinning techniques without further shearing of the fibers.

The term "truncated nanofibers" as used herein refers to nanofibers obtained by further shearing of nanofibers directly formed by electrospinning techniques.

In the vascular patch of the present invention, the inner layer, the middle layer and the outer layer are all made of biocompatible elastic polymer materials, which makes the vascular patch of the present invention have good biocompatibility and elasticity, and can be applied It is suitable for the reconstruction and repair of hearts, large blood vessels and peripheral blood vessels of various sizes; both the inner and outer layers have a porous structure formed by stacking nanofibers, and the middle layer is a dense and impermeable elastic layer formed of elastic polymer materials. The matching of the nanofiber porous structure and the dense elastic layer can make the vascular patch automatically heal the pinhole formed after the suture needle or suture thread passes through the patch, which greatly shortens the hemostasis time. Also, when the outer surface of the inner layer used to contact the site to be repaired has an anticoagulant coating, the vascular patch can effectively reduce the formation of thrombus. In addition, the loose porous structure formed by the stacking of truncated nanofibers in the outer layer facilitates the cells surrounding the vascular patch to infiltrate the patch through the loose porous structure and proliferate in the patch, accelerating the integration of the patch and autologous blood vessels. Therefore, the biocompatible vascular patch of the present invention can achieve the effects of reducing thrombosis, instantaneous hemostasis and easy matching with different repair sites.

Description of drawings

FIG. 1A is a schematic three-dimensional structural diagram of a blood vessel patch according to an embodiment of the present invention.

FIG. 1B is a cross-sectional view taken along line H-H in FIG. 1A .

Figure 2 shows the comparison of the leakage amount and the number of punctures in the anti-leakage test between the vascular patch of Figure 1 and the commercially available Gore-Tex stretch Graft patch and GoreAcuseal Patch.

Figure 3 shows the proliferation of cells cultured on the surface of a nanofibrous vascular patch according to an embodiment of the present invention, as well as cells cultured on the surface of commercially available Gore-Tex stretch Graft patches and Gore Acuseal Patch patches.

Detailed ways

The various aspects involved in the present invention will be described in detail below with reference to specific embodiments, which are only used to illustrate the present invention, and do not limit the protection scope and essential content of the present invention.

Embodiment 1. Vascular patch and preparation method thereof

As shown in FIG. 1A and FIG. 1B , the vascular patch of this embodiment includes an intermediate layer 2 and an inner layer 1 and an outer layer 3 respectively attached on opposite sides of the intermediate layer, wherein the inner layer 1 is used for contacting The site to be repaired is formed by stacking continuous nanofibers made of polylactic acid/polycaprolactone copolymer by electrospinning technology, and its outer surface is coated with a heparin anticoagulant coating by covalent grafting. The outer layer 3 has a loose porous structure formed by stacks of truncated nanofibers made of polyurethane. The intermediate layer 2 is a dense and water-impermeable polycaprolactone elastic layer.

i. Preparation of anticoagulant material-modified inner layer:

Dissolve 8g of polylactic acid/polycaprolactone copolymer (purchased from Sigma, Mn=85,000Da, the weight ratio of polylactic acid and polycaprolactone is 50:50) particles in 100ml of hexafluoroisopropanol, and configure into 8 %w/v spinning solution, electrospinning was carried out under the conditions of spinning voltage of 12kv, receiving distance of 15cm, relative humidity of 45%, room temperature, and receiving time of 8h, thereby obtaining a stack of continuous nanofibers with a thickness of For a nanofiber membrane of 100 μm, the diameter of the continuous nanofibers is 450nm±150nm, and the porosity of the obtained nanofiber membrane is 75%. The membrane was soaked in 2wt% polyethyleneimine solution at 37°C for 2h, then soaked in 0.5% glyoxal solution for 40min, washed with deionized water for 30min, repeated three times, and dried in low temperature vacuum for 24h. The dried nanofiber membrane was soaked in MES buffer solution containing 5g heparin, 2g carbodiimide, 3g hydroxysuccinimide, protected from light, treated at 37°C for 48h, rinsed with deionized water for 30min, and treated with low temperature vacuum for 24h , the outer surface of the nanofibrous membrane covalently grafted with heparin coating was obtained as the inner layer of the vascular patch.

ii. Preparation of the water-impermeable elastic interlayer

25g of polycaprolactone (Mw=80000Da) was dissolved in 100ml of dichloromethane and fully stirred for 24h, cast into a polytetrafluoroethylene mold, and dried in a low-temperature vacuum to obtain a transparent film with a thickness of 200μm as an intermediate layer.

With reference to the device in ASTM spinning 392-04 (2015) and the mold in the standard test method for bursting pressure strength in ISO7198-2017 ASTM F 2392-04, the prepared intermediate layer was subjected to bursting pressure test, taking the diameter of 4mm as above The prepared interlayer transparent diaphragm was fixed on the mold hole, and the physiological saline was injected at a rate of 2ml/min through a peristaltic pump and the pressure was monitored by a digital pressure gauge until the failure point. When the pressure reached 600mmHg, there was still no liquid leakage. leakage, which is already far greater than the systolic blood pressure of the human body of 140mmHg. This indicates that the intermediate layer can meet the requirements of being impermeable to liquids when used in the human body.

iii. Preparation of the outer layer

Dissolve 25g of polyurethane (Mw=300000Da) particles in 100ml of N,N-dimethylformamide to prepare a 25% w/v spinning solution. The spinning voltage is 14kv, the receiving distance is 15cm, and the relative humidity is 45%. , room temperature, under the condition of receiving time of 8h, a nanofiber membrane formed by stacking continuous nanofibers was obtained by electrospinning, and the nanofiber membrane was cut into small pieces with a size of 0.5*0.5mm, and then dispersed in tert-butanol Soak for 24h, then shear 30mim with a high-speed homogenizer (rotation speed 10000rpm), then, pour the dispersed and sheared nanofiber dispersion into a polytetrafluoroethylene mold, freeze-dry, and obtain a film with a thickness of 100 μm as a film with a thickness of 100 μm. An outer layer of truncated nanofibers, wherein the nanofibers have an average length of 150 μm.

iv. Preparation of Vascular Patches

The inner layer, the middle layer and the outer layer membranes prepared above were stacked in sequence, then placed in a thermocompression bonding machine, and thermobonded at a heating temperature of 85° C. and a pressure of 12 kPa for 10 min. Since the melting temperature of polycaprolactone is 65°C, the interlayer is melted and cooled again to bond the three-layer structure together to obtain a vascular patch.

Optionally, in the present invention, a two-fluid atomization spraying process can be used to coat one side of the inner layer to form an intermediate layer. Specifically, after the inner layer is prepared as described above, a solution of polycaprolactone in tetrahydrofuran (5 g of polycaprolactone (Mw=80000 Da) in 100 ml of tetrahydrofuran solution) can be used by a two-fluid atomization spray process. One side of the inner layer is coated with a dense layer with a thickness of 200 μm as an intermediate layer. Subsequently, the outer layer prepared as described above is bonded to the intermediate layer by hot pressing, thereby preparing a blood vessel patch with a three-layer structure.

Example 2. Comparison of anti-leakage effects between the nanofiber vascular patch of the present invention and commercially available products

According to the method in Section 8.3.4 of the YY0500 Artificial Vascular Standard, the nanofiber vascular patch prepared in the above-mentioned example and the commercially available vascular patch Gore-Tex stretch Graft and Gore Acuseal Patch were used under 120mmHg pressure with a 15-gauge puncture needle Repeated puncture was performed, and the number of punctures and the rate of leakage were recorded. As shown in Figure 2, the leakage rate of the commercially available vascular patches Gore-Tex stretch Graft and Gore Acuseal Patch increased significantly with the increase of the number of punctures from 20 to 60, which indicates that these two commercially available After the patch is punctured, the puncture needle hole will not heal automatically, so leakage occurs, and more and more leakage occurs due to the increase of the number of punctures. In sharp contrast, the leakage rate of the vascular patch of the present invention did not change when the number of punctures increased from 20 to 60 times, and the leakage rate was much lower than that of the commercially available vascular patch Gore- Leak speed for Tex stretch Graft and Gore Acuseal Patch. It can be seen that the vascular patch of the present invention can achieve instantaneous hemostasis at the sutured pinhole without leakage due to multiple punctures. Therefore, the pinhole will heal quickly and automatically after being sutured to the site to be repaired.

Example 3. Biocompatibility analysis of the nanofibrous vascular patch of the present invention

The cytotoxicity of the patch prepared in the above Example 1 and the commercially available vascular patch Gore-Texstretch Graft and Gore Acuseal Patch were tested by the direct contact method. The three patch materials were made into the same size and size, placed in a 24-well plate, three parallel samples in each group, and the patches were immersed in 75% ethanol for sterilization and soaked for 5 hours. After removing the ethanol, the cells were washed three times with PBS, and soaked in DMEM high-glucose medium containing 10% fetal bovine serum and 1% double antibody (penicillin/streptomycin) for 24 h. Take L929 cells in the logarithmic phase of growth, digest and pipet, and prepare a cell suspension with a concentration of 1 × 10 ⁴ cells/mL. The cell suspension is inoculated on the surface of each patch material in an amount of 200 μL per well. On the 3rd and 7th day, the cell proliferation was detected by MTT method. Use a microplate reader to detect the absorbance at the wavelength of 492nm, and the absorbance value can reflect the growth of cells. The results are shown in Figure 3. As can be seen from Figure 3, during the cell culture process from 1 to 7 days, the cells cultured on the surface of the nanofibrous vascular patch of the present invention increased with the increase of the culture days, and the cell proliferation was higher than that of the market. The vascular patch Gore-Tex stretch Graft and the Gore Acuseal Patch patch are sold, which shows that the nanofibrous vascular patch of the present invention has no cytotoxicity and has good biocompatibility.

Example 4. In vitro coagulation assay

Partial thromboplastin time (APTT) detection was performed on the patch prepared in the above Example 1 and the commercially available vascular patch Gore-Tex stretch Graft (GTSG) and Gore Acuseal Patch (GAP) patch material, prothrombin time ( PT) detection, thrombin time (TT) detection. The detection steps are as follows: add 150 μL of platelet-poor plasma to the patch material to be detected, and incubate at 37° C. for 1 h. Then, 150 μL of partial thrombin and calcium chloride solution were added respectively, and APTT was detected by automatic coagulation instrument; PT reagent was added under the same conditions, and PT was detected by automatic coagulation instrument; TT reagent was added under the same conditions, and TT was detected by automatic coagulation instrument. The coagulation test results are shown in Table 1.

Table 1. Coagulation test results of nanofibrous vascular patch of the present invention and commercially available vascular patch GTSG and GAP

As shown in Table 1 above, the coagulation time of the patch of ePTFE material without anticoagulation coating is close to that of normal blood, while the coagulation time of the nanofibrous vascular patch with heparin coating of the present invention is significantly prolonged, which shows that the present invention The anticoagulant coating in the inner layer of the nanofibrous vascular patch can significantly enhance the anticoagulant effect.

The present invention has been specifically described above with reference to the specific embodiments. These specific embodiments are only exemplary and cannot limit the protection scope of the present invention. The invention makes various modifications, changes or substitutions. Therefore, various equivalent changes made according to the present invention still belong to the scope covered by the present invention.

Claims (13)

1. A biocompatible vascular patch, comprising: an intermediate layer and an inner layer and an outer layer respectively attached to opposite sides of the intermediate layer,

wherein:

the inner layer has a porous structure formed by a stack of continuous nanofibers made of a first elastic polymer material, having a porosity of 60% to 80%;

the outer layer has a porous structure formed by a stack of truncated nanofibers having a porosity of 80% to 90%, and the nanofibers are made of a second elastic polymer material;

the intermediate layer is a water-impermeable elastic layer formed from a third elastic polymeric material.

2. The biocompatible vascular patch of claim 1, wherein the inner layer comprises an anticoagulant material.

3. The biocompatible vascular patch of claim 2, wherein the anticoagulant material is coated on an outer surface of the inner layer.

4. The biocompatible vascular patch of claim 2 or 3, wherein the anticoagulant material is heparin.

5. The biocompatible vascular patch of claim 1, wherein the nanofibers of the inner layer have a diameter of 100nm to 800nm, the nanofibers of the outer layer have a diameter of 100nm to 800nm, and the truncated nanofibers in the outer layer have a length of 60-300 μ ι η.

6. The biocompatible vascular patch of claim 1, wherein the first, second, and third elastic polymeric materials are each independently selected from the group consisting of: polylactic acid/polycaprolactone copolymers, polycaprolactone, polyurethane, polylactic acid, polylactic glycolic acid copolymers, poly (2-oxazoline), silicones, polyamide-containing polymers, polyether urethanes.

7. The biocompatible vascular patch of claim 6, wherein the first elastomeric polymer material is a polylactic acid/polycaprolactone copolymer, the second elastomeric polymer material is a polyurethane, and the third elastomeric polymer material is polycaprolactone.

8. The biocompatible vascular patch of claim 1, wherein the inner layer is formed by an electrospinning technique.

9. The biocompatible vascular patch of claim 1, wherein the outer layer is made by:

(1) preparing the second elastic polymer material into a nanofiber membrane by electrostatic spinning;

(2) shearing the nanofiber membrane prepared in the step (1), and dispersing the sheared nanofiber membrane in a solvent for homogenization treatment to obtain a nanofiber dispersion liquid;

(3) and (3) placing the nanofiber dispersion liquid in a mold with a preset size for freeze drying to obtain the outer layer with a loose porous structure formed by stacking the truncated nanofibers.

10. The biocompatible vascular patch of claim 1, wherein the intermediate layer is formed by a cast molding process, a fluid spray process, a dip coating process, an over coating process, or a vapor deposition process.

11. The biocompatible vascular patch of claim 1, wherein the inner layer has a thickness of 0.1 to 3 millimeters, the middle layer has a thickness of 0.01 to 2 millimeters, and the outer layer has a thickness of 0.05 to 3 millimeters.

12. Method for preparing a biocompatible vascular patch according to any one of claims 1 to 11, comprising the steps of:

(i) providing the inner layer, the intermediate layer and the outer layer separately and placing the inner layer and the outer layer on opposite sides of the intermediate layer;

(ii) (ii) applying a hot press to the inner, outer and intermediate layers deposited in step (i) to apply the inner and outer layers to opposite sides of the intermediate layer.

13. The method of claim 12, wherein the thermocompression bonding process is performed at a temperature of 80 to 85 ℃ and a pressure of 10 to 12 kPa.

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