CN113679890A

CN113679890A - Heart valve leaflet and preparation method thereof

Info

Publication number: CN113679890A
Application number: CN202110937381.6A
Authority: CN
Inventors: 刘婧; 王志红; 冷希岗; 孔德领
Original assignee: Institute of Biomedical Engineering of CAMS and PUMC
Current assignee: Institute of Biomedical Engineering of CAMS and PUMC
Priority date: 2021-08-16
Filing date: 2021-08-16
Publication date: 2021-11-23
Anticipated expiration: 2041-08-16
Also published as: CN113679890B

Abstract

The invention discloses a heart valve leaflet and a preparation method thereof, wherein the heart valve leaflet is of a three-layer structure which sequentially comprises a fiber layer, a sponge layer and a ventricle layer; the raw materials for preparing the fiber layer comprise a polymer and gelatin or collagen; the sponge layer is prepared from the raw materials of polymer and polysaccharide substances; the preparation raw materials of the ventricular layer comprise polymer and silk fibroin; the polymer is selected from at least one of PLCL, PLA, PLGA, PCL and PU; the heart valve leaflets are similar in composition to the components of the natural heart valve in each layer; structurally, the three-layer structure is obvious and is similar to the three-layer structure of the leaflet of the natural valve; functionally, the biological tissue culture medium has proper mechanical property, can promote cell proliferation and adhesion, maintain cell activity, can down-regulate calcification-related gene expression, shows more excellent anti-calcification, cellularization and vascularization capabilities after subcutaneous implantation, and provides a more favorable microenvironment for tissue regeneration.

Description

Heart valve leaflet and preparation method thereof

Technical Field

The invention relates to the technical field of heart valves, in particular to a heart valve leaflet and a preparation method thereof.

Background

According to the world health statistics of 2021 published by the world health organization, the morbidity and mortality of cardiovascular diseases (CVD) account for the first of all causes of human death. The artificial valve used in the present valve replacement mainly comprises two types, namely a mechanical valve and a biological valve, and has some problems: mechanical valves require lifelong anticoagulation therapy for patients due to non-physiological hemodynamics, increase the risk of patients developing major hemorrhage and thromboembolism, and are now gradually coming out of the market.

The mainstream surgical valve product in clinical use at present is a biological valve, and both the products used in transcatheter heart valve implantation (TAVR) are biological valves; the biological valve is mainly prepared from acellular bovine pericardium or porcine aortic valve crosslinked by Glutaraldehyde (GA), glutaraldehyde is the most widely applied chemical crosslinking agent for nearly 50 years, and the artificial biological valve treated by glutaraldehyde has serious late calcification, large amount of tissue matrix loss, reduced biomechanical performance and high valve leaflet decay rate, wherein the life of a patient is seriously influenced by more obvious low-age patients. In addition, free aldehyde groups generated by residual glutaraldehyde have cytotoxic effects, so that severe reactions are easily caused, and the biocompatibility of the biological valve is directly influenced. The factors cause the service life of the biological valve to be only 10-15 years, and the application of the biological valve in the sick children and the middle-aged group below 60 years is severely limited. Therefore, it is important to design and manufacture new valve materials to obtain valves that are more durable, have superior calcification resistance, are hemodynamically good, and can be organized for regeneration.

Disclosure of Invention

The invention aims to provide a heart valve leaflet and a preparation method thereof, and the heart valve material has the excellent performances of better durability, excellent calcification resistance, good hemodynamics, capability of tissue regeneration and the like.

In order to achieve the above purpose of the present invention, the following technical solutions are adopted:

the invention provides a heart valve leaflet, which is of a three-layer structure, wherein the three-layer structure sequentially comprises a fiber layer, a sponge layer and a ventricle layer;

the raw materials for preparing the fiber layer comprise a polymer and gelatin or collagen;

the sponge layer is prepared from the raw materials of polymer and polysaccharide substances;

the preparation raw materials of the ventricular layer comprise polymer and silk fibroin;

the polymer is selected from at least one of PLCL, PLA, PLGA, PCL and PU.

Preferably, the thickness of the heart valve leaflet is 300-500 μm;

the thickness of the fiber layer is 50-150 μm;

the thickness of the sponge layer is 200-400 mu m;

the thickness of the ventricular layer is 50-150 mu m.

Preferably, the mass ratio of the polymer to the gelatin or collagen in the fibrous layer is not less than 1;

the mass ratio of the polymer to the polysaccharide substance in the sponge layer is not less than 1;

the mass ratio of the polymer to the silk fibroin in the ventricular layer is not less than 1.

Preferably, the polysaccharide substance is at least one selected from hyaluronic acid, alginic acid, chitosan and cellulose;

the molecular weight of the hyaluronic acid is (0.5-200) x 10⁴。

Preferably, the collagen is selected from at least one of bovine collagen, porcine collagen, marine collagen and fish collagen.

Preferably, the silk fibroin is derived from mulberry silk or tussah silk.

In a second aspect, the present invention provides a method for preparing the above cardiac valve leaflet, which comprises the following steps:

(a) respectively dissolving preparation raw materials of a fiber layer, a sponge layer and a ventricular layer in a solvent to obtain a fiber layer solution, a sponge layer solution and a ventricular layer solution;

(b) and (3) sequentially forming a fiber layer, a sponge layer and a ventricle layer from the fiber layer solution, the sponge layer solution and the ventricle layer solution through electrostatic spinning, thus obtaining the heart valve leaflet.

Preferably, the solvent is selected from at least one of sodium hydroxide solution, dimethylformamide, formic acid and hexafluoroisopropanol.

Preferably, the concentrations of the fiber layer solution, the sponge layer solution and the ventricle layer solution are respectively 10-400 mg/mL.

Preferably, the control parameters of the electrospinning are as follows:

the flow rate of the injector is 2-20 mL/h; the voltage between the needle head and the receiver is 5-20 kV, and the distance is 5-25 cm; the rotating speed of the receiver is 100-1000 rpm;

the needle head is a blunt needle head of 20-25G.

Preferably, the preparation method further comprises the step of carrying out vacuum drying treatment on the fiber layer, the sponge layer and the ventricle layer formed by electrostatic spinning.

The heart valve leaflets consist of three layers of structures, namely a fiber layer, a sponge layer and a ventricle layer; the thickness of the natural heart valve is about 300-500 mu m, wherein the fiber layer mainly comprises collagen fibers, the fibers are arranged along the circumferential direction of the valve leaflet, and the fiber layer plays a role in stress during diastole; the sponge layer mainly comprises glycosaminoglycan and proteoglycan, and the arrangement direction is random, so that the sponge layer plays a role in buffering; the ventricular layer is made of elastic fibers, and the fibers are arranged along the radial direction of the valve leaflets to provide elasticity for the heart valve; the preparation method adopts natural materials of gelatin/collagen, polysaccharide substances, silk fibroin and polymer (such as poly L-lactic acid-epsilon-caprolactone PLCL) and utilizes an electrostatic spinning technology to prepare the bionic three-layer heart valve leaflet, wherein the polymer and Gelatin (GEL)/collagen are used for simulating a three-layer structure fiber layer, the polymer and polysaccharide substances are used for simulating a sponge layer, and the polymer and silk fibroin are used for simulating a ventricular layer; wherein, the gelatin is used as the hydrolysate of collagen and can be used for simulating the collagen of the fibrous layer; hyaluronic Acid (HA) is a macromolecular, linear glycosaminoglycan that can be used to mimic that of the sponge layer; silk Fibroin (SF) is a tough, elastic protein that can increase the elastic modulus of polymers and is used to mimic the elastic protein of the ventricular layer in the three-layer structure of a native heart valve; when the heart valve leaflet prepared by the invention is implanted in vivo, the outer fibrous layer and the ventricle layer are used for maintaining the structure and providing mechanical support, and the sponge layer in the middle layer can be controllably biodegraded to support the cells to permeate into the stent so as to enhance the secretion of extracellular matrix. The three-layer heart valve leaflet prepared by the method can ensure the biocompatibility and the mechanical property of the three-layer heart valve leaflet, and can control the fiber diameter, the porosity and the degradation rate of the stent by controlling the material mixing ratio.

Compared with the prior art, the invention has the beneficial effects that at least:

the preparation method adopts an electrostatic spinning technology to prepare the bionic three-layer heart valve leaflet, the polymer provides enough mechanical strength for the bionic three-layer heart valve leaflet, and the gelatin/collagen, the polysaccharide substance and the silk fibroin improve the biocompatibility, including promoting cell adhesion, proliferation, migration and the like, and are beneficial to the regeneration of ECM and the reconstruction of physiological functions; structurally, the bionic three-layer heart valve leaflet has an obvious three-layer structure which is similar to that of a natural valve leaflet; functionally, the bionic three-layer heart valve has proper mechanical property, can promote cell proliferation and adhesion, maintain cell activity, can reduce calcification-related gene expression, shows more excellent calcification resistance, cellularization and vascularization resistance after subcutaneous implantation, and provides a more favorable microenvironment for tissue regeneration; in addition, the bionic three-layer heart valve leaflet can promote the secretion of extracellular matrixes such as collagen, glycosaminoglycan and elastin, so that vascularization is more complete, tissue remodeling is facilitated, and the function of the bionic three-layer heart valve leaflet is more similar to that of a natural heart valve. In addition, compared with the traditional valve, the electrostatic spinning preparation process has the advantages of low cost, simplicity, high efficiency and better stability, and is beneficial to large-scale production.

Drawings

In order to more clearly illustrate the detailed description of the invention or the technical solutions in the prior art, the drawings that are needed in the detailed description of the invention or the prior art will be briefly described below. Throughout the drawings, like elements or portions are generally identified by like reference numerals. In the drawings, elements or portions are not necessarily drawn to scale.

FIG. 1 is a macro photograph and cross-sectional SEM images of Tri-layer, Mono-layer and PLCL materials in the experimental example 2 of the present invention;

FIG. 2 shows the results of mechanical property measurements of Tri-layer, Mono-layer and PLCL materials in Experimental example 2;

FIG. 3 shows the results of the platelet adhesion test in Experimental example 2 of the present invention;

FIG. 4 is a qualitative analysis result of ALP activity of 7 days and 14 days on Tri-layer, Mono-layer and PLCL of VICs cultured in the calcification-inducing medium in Experimental example 2 of the present invention;

FIG. 5 shows the process of osteoblastic differentiation and qPCR data measured by PCR in Experimental example 2;

FIG. 6 shows the results of inductively coupled plasma mass spectrometry (ICP-MS) for the quantitative determination of calcium content in Experimental example 2;

FIG. 7 shows the results of H & E staining of 4-and 8-week Wistar pups implanted subcutaneously in Experimental example 2;

FIG. 8 shows the results of immunofluorescence staining of subcutaneously implanted CD31 in 4-week and 8-week Wistar pups in Experimental example 2;

FIG. 9 shows the quantitative determination results of the number of microangioses at 4 weeks and 8 weeks in Experimental example 2 of the present invention.

Detailed Description

The following describes embodiments of the present invention in detail with reference to the following embodiments. The following examples are only for illustrating the technical solutions of the present invention more clearly, and therefore are only examples, and the protection scope of the present invention is not limited thereby.

It is to be noted that, unless otherwise specified, technical or scientific terms used herein shall have the ordinary meaning as understood by those skilled in the art to which the invention pertains.

Example 1

This embodiment is a method for preparing a heart valve leaflet, comprising the steps of:

(a) respectively dissolving PLCL/GEL, PLCL/HA and PLCL/SF in a solvent, and magnetically stirring overnight to obtain a PLCL/GEL solution, a PLCL/HA solution and a PLGL/SF solution with the final concentration of 150mg/mL, wherein the solvent is dimethylformamide; molecular weight of HA in PLCL/HA solution 200X 10⁴(ii) a The ratio of the mass of the PLCL to the mass of the GEL in the PLCL/GEL is 2; the mass ratio of the PLCL to the HA in the PLCL/HA is 2; the mass ratio of PLCL to SF in PLGL/SF is 2;

(b) filling the PLCL/GEL solution into a syringe with a 23G blunt-ended needle, placing the syringe into a syringe pump, setting the constant flow to be 6mL/h, then covering a layer of aluminum foil on a collection core rod, connecting one end of a high-voltage power supply with the needle, connecting the other end of the high-voltage power supply with a receiver, and controlling the voltage between the needle and the receiver to be 12kV and the distance to be 20 cm; the rotating speed of the receiver is 100 rpm; after a PLCL/GEL spinning layer is formed, keeping the receiver still, replacing the PLCL/GEL solution in the injector with a PLCL/HA solution, forming the PLCL/HA spinning layer on the PLCL/GEL spinning layer, and forming the PLGL/SF spinning layer on the PLCL/HA spinning layer according to the operation to obtain three layers of valve leaflets;

(c) and (4) drying the three-layer valve leaflets in vacuum for 36 hours to obtain the heart valve leaflets.

Example 2

(a) respectively dissolving PLCL/GEL, PLCL/HA and PLCL/SF in a solvent, and magnetically stirring overnight to obtain a PLCL/GEL solution, a PLCL/HA solution and a PLGL/SF solution with final concentration of 100mg/mL, wherein the solvent is formic acid; the molecular weight of HA in the PLCL/HA solution was 20X 10⁴(ii) a The ratio of the mass of the PLCL to the mass of the GEL in the PLCL/GEL is 1; the mass ratio of PLCL to HA in PLCL/HA is 1; the mass ratio of PLCL to SF in PLGL/SF is 1;

(b) filling the PLCL/GEL solution into a syringe with a 25G blunt-ended needle head, putting the syringe into a syringe pump, setting a constant flow rate at 15mL/h, then covering a layer of aluminum foil on a collection core rod, connecting one end of a high-voltage power supply with the needle head, connecting the other end of the high-voltage power supply with a receiver, and controlling the voltage between the needle head and the receiver to be 10kV and the distance to be 16 cm; the rotating speed of the receiver is 500 rpm; after a PLCL/GEL spinning layer is formed, keeping the receiver still, replacing the PLCL/GEL solution in the injector with a PLCL/HA solution, forming the PLCL/HA spinning layer on the PLCL/GEL spinning layer, and forming the PLGL/SF spinning layer on the PLCL/HA spinning layer according to the operation to obtain three layers of valve leaflets;

(c) and (4) drying the three-layer valve leaflets for 30 hours in vacuum to obtain the heart valve leaflets.

Example 3

(a) respectively dissolving PLCL/GEL, PLCL/HA and PLCL/SF in a solvent, and magnetically stirring overnight to obtain a PLCL/GEL solution, a PLCL/HA solution and a PLGL/SF solution with the final concentration of 60mg/mL, wherein the solvent is hexafluoroisopropanol; the molecular weight of HA in the PLCL/HA solution was 20X 10⁴(ii) a The ratio of the mass of the PLCL to the mass of the GEL in the PLCL/GEL is 2; the mass ratio of the PLCL to the HA in the PLCL/HA is 2; the mass ratio of PLCL to SF in PLGL/SF is 2;

(b) filling the PLCL/GEL solution into a syringe with a 20G blunt-ended needle head, putting the syringe into a syringe pump, setting the constant flow rate to be 8mL/h, then covering a layer of aluminum foil on a collection core rod, connecting one end of a high-voltage power supply with the needle head, connecting the other end of the high-voltage power supply with a receiver, and controlling the voltage between the needle head and the receiver to be 17kV and the distance to be 18 cm; the rotation speed of the receiver is 800 rpm; after a PLCL/GEL spinning layer is formed, keeping the receiver still, replacing the PLCL/GEL solution in the injector with a PLCL/HA solution, forming the PLCL/HA spinning layer on the PLCL/GEL spinning layer, and forming the PLGL/SF spinning layer on the PLCL/HA spinning layer according to the operation to obtain three layers of valve leaflets;

(c) and (4) drying the three-layer valve leaflets in vacuum for 24 hours to obtain the heart valve leaflets.

Example 4

This example is a method of preparing heart valve leaflets that is substantially the same as that of example 3, except that the PLCL is replaced with PLGA.

Example 5

This example is a method of making heart valve leaflets that is substantially the same as that of example 3, except that the PLCL is replaced with PLA.

Example 6

This example is a method of making heart valve leaflets substantially the same as in example 3, except that gelatin is replaced with bovine collagen.

Example 7

This example is a method of preparing heart valve leaflets, which is substantially the same as that of example 3, except that hyaluronic acid is replaced with chitosan.

Example 8

This example is a method of manufacturing heart valve leaflets, which is substantially the same as that of example 3, except that hyaluronic acid is replaced with cellulose.

Comparative example 1

The comparative example is a method of making a heart valve leaflet comprising the steps of:

(a) dissolving PLCL, GEL, HA and SF in solvent and magnetically treatingStirring overnight to obtain a mixed solution with the final concentration of 100mg/mL, wherein the solvent is hexafluoroisopropanol; the molecular weight of HA in the mixed solution is 20X 10⁴(ii) a The ratio of the mass of the PLCL to the mass of the GEL is 2; the ratio of the mass of PLCL to the mass of HA is 2; the mass ratio of PLCL to SF is 2;

(b) filling the mixed solution into a syringe with a 20G blunt-ended needle head, filling the syringe into a syringe pump, setting the constant flow rate to be 8mL/h, then covering a layer of aluminum foil on a collection core rod, connecting one end of a high-voltage power supply with the needle head, connecting the other end of the high-voltage power supply with a receiver, controlling the voltage between the needle head and the receiver to be 17kV, and controlling the distance to be 18 cm; the rotation speed of the receiver is 800 rpm; forming a spun layer to obtain a single-layer valve leaflet;

(c) and (4) carrying out vacuum drying on the single-layer valve leaflets for 24 hours to obtain the heart valve leaflets.

Comparative example 2

(a) dissolving PLCL in a solvent and magnetically stirring overnight to obtain a mixed solution, wherein the solvent is hexafluoroisopropanol; the concentration of PLCL in the mixed solution is 100 mg/mL;

Experimental example 1

This example is a study of the effect of HA molecular weight and HA content in PLCL/HA on the proliferation of Human Umbilical Vein Endothelial Cells (HUVEC)

HA was selected to have a relative molecular weight of 20X 10⁴The PLCL/HA is dissolved in Formic Acid (FA) solvent, the concentration of the solution is 180mg/mL, and the mass ratio of the PLCL to the HA is 1: 1. the prepared PLCThe L/HA solution was filled into a 10mL syringe fitted with a 23G blunt tip needle. The syringe was loaded into a syringe pump and a constant flow rate of 6mL/h was set. One end of the high-voltage power supply is connected with the needle head, and the other end of the high-voltage power supply is connected with the receiver. Preparing heart valve leaflets by using the voltage between the needle head and the receiver as 12kV, the distance as 20cm and the rotating speed of the receiver as 100 rpm;

heart valve leaflets with HA relative molecular weights of 0.5 ten thousand, 1 ten thousand, 10 ten thousand, 120 ten thousand and 200 ten thousand and HA concentrations of 10, 100, 200, 300 and 500 mug/mL in the PLCL/HA solution were prepared according to the above method, respectively;

the experimental method comprises (a) sterilizing the PLCL/HA electrostatic spinning bracket. (b) Cutting the material to 1 × 1cm, placing the material in a culture medium at a concentration of 0.2g/mL according to ISO 10993-12:2012, and culturing at 37 deg.C with 5% (v/v) CO₂Leaching for 24 hours in an incubator, and filtering by using a sterile filter membrane of 0.22 mu m to obtain a PLCL/HA electrostatic spinning support leaching liquor. (c) HUVECs were collected, plated at 3000 cells/well in 96-well plates at 37 ℃ with 5% (v/v) CO₂Culturing in an incubator. (d) After 24 hours, the medium was aspirated and 100. mu.L of PLCL/HA electrospun scaffold leach solution was added to each well. (e) After 1, 3, 5 days, the medium was aspirated and washed 3 times with sterile PBS. (f) 10 μ L of CCK-8 serum-free medium was added to all wells and incubated at 37 ℃ for 2 hours in the absence of light. Measuring OD value at 450nm by using an enzyme-labeling instrument;

the experimental results are as follows: the effect of HA on HUVEC proliferation exhibits both size-dependent and concentration-dependent properties. On the one hand, the effect on the proliferation of HUVEC is more obvious as the molecular weight of HA is reduced, namely, the low molecular weight HA can promote the proliferation of HUVEC more. On the other hand, the effect on HUVEC proliferation shows a trend of increasing firstly and then decreasing with the increase of the HA concentration, the effect on HUVEC proliferation shows an increasing trend within the concentration range of 10-100 mu g/mL, and shows a decreasing trend within the range of 100-500 mu g/mL, when the HA concentration is 100 mu g/mL, the effect of promoting proliferation is most obvious, and the effect of promoting cell proliferation cannot be continuously enhanced by continuously increasing the concentration.

Experimental example 2

Respectively preparing heart valve leaflets according to the preparation methods of the embodiment 3 and the comparative examples 1-2, wherein the heart valve leaflet prepared in the embodiment 3 is named as Tri-layer, and the heart valve leaflet prepared in the comparative example 1 is named as Mono-layer; the heart valve leaflets prepared in comparative example 2 were pure PLCL groups;

1. scanning the cross sections of the Tri-layer, the Mono-layer and the pure PLCL by an electron microscope and taking a macro picture, wherein the scanning and taking results are shown in figure 1, in the figure, a, b and c are corresponding SEM pictures, and d, e and f are corresponding macro pictures;

as can be seen from FIG. 1, the mean fiber diameter of the Tri-layer group varied between 3.17 and 4.66 μm; wherein the PLCL/SF layer is 3.43-4.28 μm, and the PLCL/GEL layer is 3.17-4.66 μm.

2. The mechanical properties of the Tri-layer, Mono-layer and pure PLCL are tested, each sample is cut into 3 x 1cm, the thickness of the PLCL/GEL electrospun scaffold is measured by a thickness meter, stretching is carried out at the speed of 10mm/min until the material is broken, and the stress-strain curve under the load of 1kN is recorded. And calculating parameters such as ultimate tensile strength, elongation at break, elastic modulus and the like of the test sample from the stress-strain curve graph. The results of the measurement are shown in FIG. 2, in which a is the elastic modulus, b is the ultimate tensile strength, and c is the elongation at break. NS means no significant difference, p <0.05, p < 0.01;

as can be seen from FIG. 2, compared with the simple PLCL, the elastic modulus of the Tri-layer is enhanced (32.18 +/-4.19 MPa), the maximum stress is increased (10.73 +/-1.39 MPa), the elongation at break is reduced (153.95 +/-7.82%), namely after SF, HA and GEL are added into the PLCL, the mechanical property is obviously improved, and the mechanical property of the Tri-layer group is superior to that of the Mono-layer group.

3. Collagenase enzymolysis experiment is carried out on the Tri-layer, Mono-layer and pure PLCL

50mL of collagenase solution containing 302.85mg of Tris and 5.55mg of CaCl is prepared by deionized water₂，10mg NaN₃2mg of collagenase, which is filtered and sterilized by a sterile filter membrane of 0.22 mu m for later use, and the pH value is adjusted to 7.8 by hydrochloric acid; drying Tri-layer, Mono-layer and pure PLCL fully, cutting into 1 × 1 cm; weigh and record the original weight as M0. The sample is put into a centrifuge tube,adding collagenase solution, placing on a shaking table, and shaking at room temperature; and after 0-12 weeks, taking out the bionic three-layer valve leaflets, washing the bionic three-layer valve leaflets with deionized water for 3 times, and drying the bionic three-layer valve leaflets in a constant-temperature drying box at the temperature of 80 ℃ until the weight is constant. Again weighed and the weight recorded was M1. The weight loss ratio was calculated using the following formula: the weight loss rate (%) - (M1-M0)/M0 × 100%;

the results show that the collagenase enzymolysis rate of the Tri-layer valve leaflet Tri-layer group is lower than that of the Mono-layer group, but no significant difference exists between the Tri-layer group, the Mono-layer group and the pure PLCL group.

4. Blood compatibility test of the above Tri-layer, Mono-layer and pure PLCL

Platelet adhesion assay: the Tri-layer, Mono-layer and pure PLCL were cut into a size of 24-well plate using a punch, washed once with physiological saline, blotted on filter paper, placed in a 24-well plate, and spread flat (n ═ 5). The rabbit whole blood containing the anticoagulant was centrifuged at 3000rpm for 15 minutes, and the supernatant was slowly aspirated to obtain high-concentration platelet plasma (PRP). To each well was added 0.3mL of PRP and incubated at 37 ℃ for 60 minutes. Rinsed three times with sterile PBS for 3 minutes each. Fixation with 2.5% (v/v) glutaraldehyde was carried out at room temperature for 60 minutes. Dehydrating with 70%, 80%, 90%, and 100% ethanol gradient. Observing the number and state of platelets adhered to the surface of the material by using an SEM (scanning Electron microscope); the observation results are shown in FIG. 3;

as can be seen from fig. 3: the results of platelet adhesion experiments show that no platelets adhere to the surfaces of the three groups of materials.

Measuring hemolysis rate: tri-layer, Mono-layer and pure PLCL were cut to 1X 1cm (n. times.5) and incubated in 10mL of physiological saline at 37 ℃ for 10 min. 8mL of rabbit whole blood containing an anticoagulant was added to 10mL of physiological saline to obtain 18mL of diluted blood. To a sample of 10mL of physiological saline, 0.2mL of diluted blood was added and incubated at 37 ℃ for 60 minutes. Centrifuge at 1000g for 10 min. 100. mu.L of the supernatant was added to a 96-well plate, and the OD value was measured at 545nm using a microplate reader. ODs represent the OD values of the experimental groups (Tri-layer, Mono-layer or pure PLCL + diluted blood), ODp represents the OD value of the positive control group (deionized water + diluted blood), and ODn represents the OD value of the negative control group (normal saline + diluted blood). The hemolysis ratio (%) - (ODs-ODn)/(ODp-ODn) × 100%.

The result of calculation is that the hemolysis rate of the three groups of materials is lower than 0.1 percent and far lower than the national hemolysis rate standard (GB/T16886);

from the above results, it can be seen that: no obvious difference is found among the three groups, but the hemolysis rate of the Tri-layer valve leaflet group is the lowest, which indicates that the blood compatibility of the bionic Tri-layer valve leaflet is the best.

5. In vitro osteogenic differentiation assay for valve stromal cells (VICs) on Tri-layers, Mono-layers, and pure PLCL as described above

After culturing the cells for 24 hours, the ordinary low-sugar DMEM medium was discarded, and the ordinary low-sugar DMEM medium was added to the negative control group (N.C.), and the calcification-inducing medium was added to the positive control group (P.C.) and other experimental groups, and the incubation was continued at 37 ℃ and 5% (v/v) CO₂Culturing in an incubator. The calcification induction medium was changed every other day. After 7 and 14 days of incubation with calcification-inducing medium, the medium was aspirated and the material was washed with sterile PBS. Detecting the osteogenic differentiation degree of the cells through an alkaline phosphatase (ALP) quantitative qualitative experiment and a quantitative polymerase chain reaction (qPCR) experiment, thereby determining the calcification level of the cells; qualitative analysis results of ALP activity of VICs cultured in the calcification induction medium on Tri-layer, Mono-layer and pure PLCL for 7 days and 14 days are shown in FIG. 4, and PCR (polymerase chain reaction) experiments for detecting osteogenic differentiation process of cells and qPCR (quantitative polymerase chain reaction) data are shown in FIG. 5, wherein a is involved in the gene expression of osteogenic differentiation; b is Twist1, c is RUNX2, d is ATF4, e is ALP, f is OSX, g is OCN, h is OPN and i is RANKL gene expression level; denotes p<0.05, represents p<0.01, represents p<0.001 denotes p<0.0001；

As can be seen from fig. 4 and 5, the qualitative staining result of in vitro alkaline phosphatase (ALP) showed that the positive control group (p.c. group) stained significantly positive for ALP with the highest amount; while the negative control group (n.c. group) showed no significant positive appearance. Calcified nodules of VICs on the Tri-layer were significantly reduced compared to the positive control group on both days 7 and 14. As can be seen from the qPCR results, all three groups of materials can significantly down-regulate the expression of the osteogenic differentiation related genes at day 7 and 14, compared to the positive control group. The Mono-layer material has no significant difference with the Tri-layer material in the degree of down-regulating Twist1, ALP, ATF4, OCN and other related genes, which shows that under the condition of consistent composition proportion, the structural difference has little influence on calcification. Furthermore, Tri-layer was more pronounced at down-regulating RUNX2, OSX, OPN and RANKL genes than the PLCL group, suggesting that Tri-layer can delay the onset of calcification by the above-mentioned pathway.

6. Evaluation of the Tri-layer, Mono-layer and pure PLCL Material calcification in subcutaneous implant model

The experimental animals selected about 50g of Wistar male young mice, the Tri-layer, Mono-layer and pure PLCL material were cut into 1X 1cm size, 10% (w/v) chloral hydrate anesthetized rats (0.33mL/100g body weight) were injected into the abdominal cavity, and then the skin was shaved. Respectively implanting an experimental group sample and a glutaraldehyde crosslinking control group sample (GA-BHV) into the left and right sides of the back subcutaneously, suturing a skin incision, euthanizing the animal after 4-8 weeks, and taking out a graft;

drying the taken materials in a constant-temperature drying box at 80 ℃ to constant weight, weighing, and recording the weight for microwave digestion of nitric acid. After digestion, calcium content was quantitatively measured by inductively coupled plasma mass spectrometry (ICP-MS), and the measurement results are shown in fig. 6, where p is <0.05, p is <0.01, and p is < 0.001.

Histological staining qualitative observation of calcification: hematoxylin & eosin (H & E) and Von Kossa staining visually reflected the histomorphological changes and local microcalcification distribution of the samples, with H & E staining of 4 weeks (a-c) and 8 weeks (d-f) Wistar pups implanted subcutaneously as shown in fig. 7 at a scale bar of 50 μm;

immunofluorescent staining of subcutaneously implanted CD31 in 4-week (a-c) and 8-week (d-f) Wistar pups is shown in FIG. 8, where microvessels are labeled with CD31 antibody (red fluorescence) and nuclei are labeled with DAPI (blue fluorescence). Scale bar 50 μm;

the results of quantitative determination of the number of microvessels at 4 and 8 weeks are shown in fig. 9, where p <0.001 and p < 0.0001.

As can be seen from FIG. 6, the calcium ion content of Tri-layer was as low as 0.13. + -. 0.03. mu.g/mg at week 4, and the value at week 8 was 0.19. + -. 0.04. mu.g/mg, both of which were significantly lower than those of PLCL and Mono-layer groups, indicating that Tri-layer had better anti-calcification ability. Furthermore, the calcification level of Tri-layer was much lower than that of glutaraldehyde cross-linked biological heart valve (GA-BHV) (21.8. + -. 32.6. mu.g/mg) used clinically, and also lower than that of commercial Sino product (0.7. + -. 1.2. mu.g/mg). The results show that in a subcutaneous implantation experiment, the calcification resistance of the Tri-layer is obviously superior to that of GA-BHV, and the biggest defect of GA-BHV is overcome.

As can be seen from FIG. 7, the Tri-layer, Mono-layer and pure PLCL materials maintained a monolithic structure, and cells infiltrated from the outer surface to the interior of the Tri-layer, Mono-layer and pure PLCL materials. The cells infiltrated by the PLCL group are only concentrated at the marginal part, and the number of infiltrated cells is small. After 8 weeks of transplantation, a well-regenerated layer of new tissue was visible by both Mono-layer and Tri-layer, with an average thickness greater than that of the PLCL control. And the Tri-layer has better biocompatibility, so cells almost infiltrate the whole scaffold, the depth is deeper, the distribution is more uniform, and the cellularization degree is more complete. Therefore, the Mono-layer and Tri-layer groups added with GEL, HA and SF have better biocompatibility and are beneficial to cell infiltration; secondly, although the Mono-layer and Tri-layer groups both contain HA with the same HA content, in the Tri-layer, PLCL + HA is located in the middle layer, and forms a certain concentration gradient with the upper and lower layers after being embedded subcutaneously, the middle layer HAs high concentration, and the outer side HAs low concentration, so that the HA located in the middle layer can play a chemotactic role, and cells on the outer side can more easily migrate to the middle part of the Tri-layer.

As can be seen from fig. 8 and 9, at week 4 and 8, the number of microvasculature in the PLCL group is the least and mainly distributed around the outside of the stent, while the number of microvasculature in the Tri-layer is the most and uniformly distributed throughout the three-layered valve; FIG. 9 quantification results also indicate that the number of neoformed microvessels in the Tri-layer is significantly higher than in the PLCL control group. At week 8, the number of new microvessels increased with time, specifically, the PLCL group: 14.14 +/-2.11; mono-layer group: 33.94 +/-4.16; tri-layer group: 54.14 + -2.67. The capillaries formed in the stent are beneficial to transporting oxygen and nutrient substances for growing cells, and meanwhile, metabolic waste can be transported away, thereby being beneficial to tissue regeneration.

In conclusion, the Tri-layer has better mechanical property and stability, can promote cell proliferation and adhesion, maintain cell activity, can reduce calcification-related gene expression, shows more excellent calcification-resistant, cellularization and vascularization capabilities after subcutaneous implantation, and provides a more favorable microenvironment for tissue regeneration.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; such modifications and substitutions do not depart from the spirit and scope of the present invention, and they should be construed as being included in the following claims and description.

Claims

1. A heart valve leaflet is characterized in that the heart valve leaflet is of a three-layer structure, and the three-layer structure sequentially comprises a fiber layer, a sponge layer and a ventricle layer;

the polymer is selected from at least one of PLCL, PLA, PLGA, PCL and PU.

2. The heart valve leaflet of claim 1, wherein the heart valve leaflet has a thickness of 300-500 μ ι η;

the thickness of the fiber layer is 50-150 μm;

the thickness of the sponge layer is 200-400 mu m;

the thickness of the ventricular layer is 50-150 mu m.

3. A heart valve leaflet according to claim 1 or 2, characterized in that the mass ratio of polymer to gelatin or collagen in the fibrous layer is not less than 1;

4. The heart valve leaflet of claim 1, wherein the polysaccharide substance is selected from at least one of hyaluronic acid, alginic acid, chitosan, and cellulose;

the molecular weight of the hyaluronic acid is (0.5-200) x 10⁴。

5. A heart valve leaflet as claimed in claim 1 wherein the collagen is selected from at least one of bovine collagen, porcine collagen, marine collagen and fish collagen.

6. The heart valve leaflet of claim 1, wherein the silk fibroin is derived from mulberry silk or tussah silk.

7. The method of making a heart valve leaflet as claimed in any of claims 1 to 6, characterized in that it comprises the steps of:

8. The production method according to claim 7, wherein the solvent is at least one selected from the group consisting of sodium hydroxide solution, dimethylformamide, formic acid and hexafluoroisopropanol.

9. The method according to claim 1, wherein the concentrations of the fiber layer solution, the sponge layer solution and the ventricular layer solution are 10 to 400mg/mL, respectively.

10. The method according to claim 1, wherein the control parameters of the electrospinning are as follows:

the needle head is a blunt needle head of 20-25G.