CN116115825B

CN116115825B - Preparation method of conjunctiva stent, conjunctiva stent and application

Info

Publication number: CN116115825B
Application number: CN202211371070.9A
Authority: CN
Inventors: 薛佳佳; 龚博文; 张馨丹
Original assignee: Beijing University of Chemical Technology
Current assignee: Beijing University of Chemical Technology
Priority date: 2021-11-15
Filing date: 2022-11-03
Publication date: 2024-12-24
Anticipated expiration: 2042-11-03
Also published as: CN116115825A

Abstract

The invention provides a preparation method of an eye conjunctiva stent, an eye conjunctiva stent and application thereof. The method comprises the steps of adding a high polymer into a solvent A, dissolving to obtain a solution A, carrying out electrostatic spinning on the solution A by using a roller as a receiver to obtain a fiber film with a single-axis ordered structure, adding a natural high polymer A into a solvent B, dissolving to obtain a solution B, adding a growth factor into a solvent C, dissolving to obtain a solution C, carrying out coaxial electrostatic spraying on the fiber film with the single-axis ordered structure and the solution C respectively serving as a shell layer and a core layer by using the fiber film with the single-axis ordered structure as the receiver, or carrying out single-axis electrostatic spraying on the mixed solution B and the mixed solution C, so as to obtain the eye conjunctiva stent. The ocular conjunctiva stent provided by the invention has the effects of inducing directional migration of epithelial cells and promoting cell growth, can accelerate conjunctival regeneration, and can be degraded and replaced by nascent conjunctiva or degraded and separated from the surface of conjunctiva after healing.

Description

Preparation method of conjunctiva stent, conjunctiva stent and application

Technical Field

The invention relates to the technical field of biological materials, in particular to a preparation method of an eye conjunctiva stent, an eye conjunctiva stent and application thereof.

Background

The conjunctiva of the eye is a layer of translucent mucosal tissue that begins at the end of the limbic margin and covers the posterior eyelid and the anterior segment sclera surface. When the conjunctiva of the eye is damaged by various trauma, thermal burns, chemical burns or severe ocular surface diseases, defects in conjunctival tissue may result. Autologous conjunctival transplantation, allogeneic conjunctival transplantation or amniotic membrane transplantation is now commonly used clinically to repair conjunctival defects. The normal conjunctiva that can be used for autograft is limited and is a method of 'removing east wall and repairing west wall', which can cause damage to itself. Normal conjunctiva, which can be used for allograft, also faces material shortage problems and risks immune rejection. Amniotic membrane transplantation also has disadvantages. In an ocular inflammatory environment, the amniotic membrane can be rapidly degraded, and the growth of epithelial cells cannot be effectively guaranteed for a long time. In addition, individual differences in amniotic donor make the thickness and quality of amniotic membrane grafts different, with the risk of infection by infectious disease.

Therefore, in order to solve the problems of insufficient autologous and allogeneic conjunctiva transplantation donors and too fast amniotic membrane transplantation degradation rate, the development of an artificial conjunctiva stent which is suitable in degradation time and can accelerate the repair of conjunctival defects of eyes has great significance.

Disclosure of Invention

In order to solve the technical problems in the prior art, the invention provides a preparation method of an eye conjunctiva stent, the eye conjunctiva stent and application.

The artificial conjunctiva stent provided by the invention has the advantages that the degradation time is proper, the repair of conjunctival defects of eyes can be quickened, the conjunctiva defects of eyes can be sewed at the damaged position after being damaged, the repair of the conjunctiva is quickened, and the conjunctiva is degraded after being repaired.

The invention constructs electrostatic spinning fiber with a single-axis ordered arrangement structure as an outer layer, combined active particles as an inner layer (the inner layer is an electrostatic spray layer loaded with an active particle core-shell structure, the core layer contains growth factors, and the shell layer is natural high polymer), and the like, so as to continuously release required active particles from an eye conjunctiva stent, wherein the outer layer is the electrostatic spinning fiber with the single-axis ordered arrangement structure, plays a role in guiding directional migration and proliferation of epithelial cells, and the active particles containing the growth factors play a role in promoting growth of the epithelial cells, so that the eye conjunctiva stent can be degraded and replaced by new eye conjunctiva, or can be degraded and separated on the surface of the eye conjunctiva after healing.

The conjunctiva stent is not a single synthetic high molecular polymer, but a synthetic high molecular polymer or a natural high molecular polymer and a synthetic high molecular polymer so as to improve the biocompatibility of the conjunctiva stent, control the degradation rate and load active particles so as to promote the repair rate of conjunctiva injury.

The invention aims at providing a preparation method of an eye conjunctiva stent, which comprises the following steps:

(1) Adding a high molecular polymer into the solvent A, and dissolving to obtain a solution A;

(2) Carrying out electrostatic spinning on the solution A obtained in the step (1) by using a roller as a receiver to obtain a fiber membrane with a uniaxial ordered arrangement structure;

(3) Adding the natural high polymer A into the solvent B, dissolving to obtain a solution B, adding the growth factor into the solvent C, and dissolving to obtain a solution C;

(4) And (3) taking the fiber membrane with the uniaxial ordered structure obtained in the step (2) as a receiver, and respectively taking the solution B and the solution C as a shell layer and a core layer to carry out coaxial electrostatic spraying, or mixing the solution B and the solution C to carry out uniaxial electrostatic spraying, so as to obtain the conjunctival stent.

In a preferred embodiment of the present invention,

Step (1),

The high molecular polymer is a mixture of a natural high molecular polymer B and a degradable synthetic high molecular polymer or the degradable synthetic high molecular polymer, wherein the degradable synthetic high molecular polymer is preferably at least one of polylactic acid, polycaprolactone, polylactic acid-glycolic acid copolymer and polylactic acid-glycolic acid-caprolactone copolymer;

The solvent A is at least one of hexafluoroisopropanol, trifluoroethanol, chloroform, methanol, dichloromethane, acetic acid and aqueous solution thereof, and N, N' -dimethylformamide.

In a preferred embodiment of the present invention,

The mass ratio of the degradable synthetic high molecular polymer to the natural high molecular polymer in the mixture of the natural high molecular polymer B and the degradable synthetic high molecular polymer is less than 100, and the mass ratio is preferably (0.1-10): 1;

the mass concentration of the solution A is 5-24%, and preferably 6-12%.

In a preferred embodiment of the present invention,

Step (2),

The pushing rate of the solution A is 0.1-10 mL/h, preferably 1-2 mL/h;

The voltage is 8-30 kV, preferably 15-25 kV;

The receiving distance is 10-25 cm, preferably 15-20 cm;

The rotating speed of the roller adopts a mode of firstly high speed and then low speed:

the initial drum rotation speed is 800-3000 rpm, preferably 1000-2000 rpm, more preferably 1000-1500 rpm, and the spinning time at the high rotation speed is 10-480 min, preferably 30-120 min, more preferably 40-60 min;

The rotation speed of the roller is then reduced to 10-200 rpm, preferably 10-100 rpm, and the spinning time at the low rotation speed is 10-480 min, preferably 30-120 min, more preferably 40-60 min.

The electrostatic spinning fiber can be collected by a roller collector rotating at a high speed (more than 800 rpm), so that the fiber in single-axis ordered arrangement can be obtained, the single-axis ordered arrangement is favorable for directional migration and growth of cells, conjunctiva repair is hopeful to be quickened, and the fiber in random arrangement can be obtained by collecting by a roller collector rotating at a low speed (less than 200 rpm), so that mechanical strength is provided for materials.

In a preferred embodiment of the present invention,

Step (3),

The natural high molecular polymer A is at least one of collagen, gelatin, chitosan, starch, cellulose and elastin;

the solvent B is at least one of hydrochloric acid, acetic acid, ethyl acetate, glycerol, trifluoroethanol and hexafluoroisopropanol;

The solvent C is at least one of physiological saline, ultrapure water, buffer solution, methanol and ethanol;

The growth factor is at least one of an epidermal growth factor, a nerve growth factor, a vascular endothelial growth factor, a platelet derived growth factor and an acidic fibroblast growth factor.

In a preferred embodiment of the present invention,

Step (3),

The concentration of the solution B is 1 mg/mL-200 mg/mL, preferably 10 mg/mL-50 mg/mL, and more preferably 20 mg/mL-40 mg/mL;

the concentration of the solution C is 10 mu g/mL-10 mg/mL, preferably 50 mu g/mL-5 mg/mL, and more preferably 50 mu g/mL-2 mg/mL.

In a preferred embodiment of the present invention,

Step (4),

The dosage ratio of the fiber film with the uniaxially ordered structure to the total volume of the solution B and the solution C is 1g of (1-60) mL of solution, preferably 1g of (5-20) mL of solution;

In the coaxial electrostatic spraying process, the volume ratio of the solution B to the solution C is 1 (0.1-1), and preferably 1 (0.2-0.4);

In the case of uniaxial electrostatic spraying, the volume ratio of the solution B to the solution C is 1 (0.1 to 10), preferably 1 (0.3 to 3).

In a preferred embodiment of the present invention,

Step (4),

In the coaxial electrostatic spraying process, the pushing rate of the solution B electrospraying solution is 0.1-10 mL/h, preferably 1-3 mL/h, the pushing rate of the solution C electrospraying solution is 0.1-5 mL/h, preferably 0.4-1 mL/h, the voltage is 8-30 kV, preferably 15-25 kV, the receiving distance is 8-30 cm, preferably 15-20 cm, and the spraying time is 5-360 min, preferably 15-120 min, more preferably 15-40 min;

In the single-axis electrostatic spraying process, the advancing rate of the electrospray solution after the solution B and the solution C are mixed is 0.5-10 mL/h, preferably 0.5-3 mL/h, the voltage is 8-30 kV, preferably 15-25V, the receiving distance is 8-30 cm, preferably 10-25 cm, and the spraying time is 5-360 min, preferably 15-120 min, and more preferably 15-60 min.

The concentration and the propelling speed of the solution are adjusted to be matched, so that the amount of nano particles deposited on the fiber membrane is adjusted, the solution B wraps the solution C when in coaxial electrostatic spraying, the slow release effect on the growth factors is realized, the single axis is the mixture of two substances, the release of the growth factors is faster, the slow release of the growth factors is required when the injury is large, the ophthalmic conjunctiva stent prepared by coaxial electrostatic spraying can be used, the fast release of the factors is required when the injury is small, and the ophthalmic conjunctiva stent prepared by single axis electrostatic spraying can be used.

Another object of the present invention is to provide an conjunctival stent.

The eye conjunctiva stent is of a two-layer structure, the outer layer is an electrostatic spinning fiber of a uniaxial ordered arrangement structure, and the inner layer is an electrostatic spraying layer loaded with active particles.

The conjunctiva support can be cut into any size according to actual needs, and the length range is 3 mm-30 mm, and the width range is 2 mm-20 mm according to clinical general needs.

The invention further aims to provide an application of the conjunctival stent in repairing conjunctival injury.

The invention adopts the following technical scheme:

the preparation method specifically comprises the following steps:

Step (1), adding a degradable synthetic high molecular polymer or a natural high molecular polymer and a degradable synthetic high molecular polymer into a solvent A, and fully dissolving to obtain a solution A;

step (2), carrying out electrostatic spinning on the solution A by using a roller rotating at a high speed as a receiver to obtain a fiber membrane with a single-axis ordered arrangement structure;

Step (3), adding the natural high molecular polymer into the solvent B to obtain a solution B, and adding the growth factor into the solvent C to obtain a solution C;

And (4) taking the uniaxially ordered fiber membrane obtained in the step (2) as a receiver, respectively taking the solution B and the solution C as a shell layer and a core layer for coaxial electrostatic spraying, or mixing the solution B and the solution C for uniaxial electrostatic spraying to obtain the active particle loaded uniaxially ordered fiber membrane, and preparing the active particle loaded conjunctiva support.

The nanofiber membrane obtained by electrostatic spraying is still required to be placed in a fume hood for 2-3 days to remove the solvent.

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

the artificially synthesized conjunctiva support provided by the invention plays a role in repairing membranes, solves the problem of insufficient donor of autologous or allogenic conjunctiva transplantation, and simultaneously solves the problem of excessively rapid degradation in amniotic membrane repair by adopting a polymer with adjustable degradation rate.

The conjunctiva stent is prepared by an electrostatic spraying mode, nano particles are sprayed onto fibers in the form of liquid drops during spraying, the liquid drops are bonded with the fibers, the nano particles formed after drying have stronger bonding force with the fibers, only the conjunctiva injury facing side exists, and the particle load can be changed by adjusting the concentration of the solution B and the solution C and the relevant parameters of electrostatic spraying.

According to the eye conjunctiva support prepared by the invention, the electrostatic spinning fiber is collected through the roller collector rotating at a high speed (more than 800 rpm), so that the fiber which is in single-axis ordered arrangement can be obtained, and compared with a random electrostatic spinning fiber film, the fiber which is in ordered single-axis ordered arrangement can recruit cells through a surface contact induction effect, the natural high molecular polymer in the active particles is beneficial to protecting the growth factors, meanwhile, a specific coarse structure or a patterning structure is constructed on the surface of the eye conjunctiva support, the migration of the cells can be induced, the loaded growth factors can promote the growth of the cells, and the repairing effect of conjunctival injury is improved. Whereas collection by a low speed rotating (less than 200 rpm) roller collector results in a random arrangement of fibers, this side does not directly contact the conjunctival wound, providing better mechanical strength to the film material.

The conjunctiva support provided by the invention can be cut into different sizes to match the size of damaged conjunctiva, so that the repairing effect is improved.

Drawings

FIG. 1 is a schematic view of an conjunctival stent;

1 is a uniaxial ordered array of fibers, 2 is electrostatically sprayed particles;

FIG. 2 is an SEM image of the uniaxial ordered array of polycaprolactone obtained in step (2) of example 1;

FIG. 3 is a graph showing the results of cell proliferation experiments of the conjunctival stent prepared in example 1;

FIG. 4 is an SEM image of an conjunctival stent obtained in example 2;

FIG. 5 is a migration chart of fibroblasts in the conjunctival ocular stent obtained in example 3;

FIG. 6 is an SEM image of polycaprolactone-chitosan fiber obtained in example 1, step (2) of example 4;

Fig. 7 is an SEM image of the conjunctival stent obtained in example 5.

Detailed Description

The present invention is described in detail below with reference to the specific drawings and examples, and it is necessary to point out that the following examples are given for further illustration of the present invention only and are not to be construed as limiting the scope of the present invention, since numerous insubstantial modifications and adaptations of the invention to those skilled in the art will still fall within the scope of the present invention.

The starting materials used in the examples were all conventional commercially available.

The testing method comprises the following steps:

Proliferation of cells was examined by CCK-8 method. Cutting the sterilized material into material with diameter of 1.5cm, spreading on the bottom of 24-well plate, collecting L929 grown to the bottom of the culture flask, transferring cell suspension into the well plate at 100 μl/well, seeding on blank hole and material surface, adding 900 μl of cell culture solution into each hole, and placing the 24-well plate into cell incubator. CCK-8 was tested at 1 day, 3 days, and 5 days, and after the original culture broth was aspirated from each well, a CCK-8 solution was prepared by mixing the cell culture broth with 10% by volume of the CCK-8 reagent, and was added thereto at 500. Mu.L/well. The absorbance at a wavelength of 450nm was measured after placing the well plate in the cell incubator for 4 hours.

Fibroblast migration ocular conjunctival stents were washed three times with cell culture medium prior to fibroblast seeding. The sterilized polydimethylsiloxane strips were then placed on the left side of the sample leaving a 0.5cm wide area to be seeded into fibroblasts. After 4 hours of cell adhesion, the strips were removed to allow the cells to migrate forward. After three more days of incubation, the cells were stained separately. Cells were fixed in 3% glutaraldehyde solution for 10min, permeabilized with 0.1% Triton X-100 for 5min, and then blocked with 3% BSA solution for 1 hr at room temperature. Then immersed in a solution of Alexa Fluor 555 phalloidin and 4', 6-diamidine-2-phenylindole, respectively, at room temperature for 20 and 5min. After each run, the cells were washed three times with PBS solution. After staining, the samples were photographed under a confocal laser scanning microscope.

Example 1

Step (1), dissolving polycaprolactone in dichloromethane, and magnetically stirring at room temperature for 12 hours to obtain a solution A with the mass concentration of 10%;

Step (2), carrying out electrostatic spinning by using the solution A, wherein when a receiver of the electrostatic spinning is a roller with the rotating speed of 1000rpm, the advancing speed of the solution A is 1ml/h, the voltage is 15kV, the receiving distance is 20cm, the spinning is carried out for 60min, the rotating speed is reduced to 10rpm, other spinning conditions are unchanged, and the spinning is carried out for 40min, so as to obtain the polycaprolactone fiber membrane with the single-axis ordered arrangement structure;

Step (3), dissolving collagen in acetic acid aqueous solution, magnetically stirring at room temperature for 12 hours, fully mixing to obtain 20mg/mL shell solution B, loading epidermal growth factor, and dissolving in physiological saline to obtain 1mg/mL nuclear layer solution C;

And (4) spraying coaxial static electricity on the polycaprolactone fiber with the outer layer single-axis ordered structure obtained in the step (2), wherein the dosage ratio of the fiber film with the single-axis ordered structure to the total volume of the solution B and the solution C is 1g of the fiber film with the single-axis ordered structure, 18mL of the solution is adopted, the advancing rate of the shell electrospraying solution is 3mL/h, the advancing rate of the core electrospraying solution is 1mL/h, the voltage is 22kV, the receiving distance is 18cm, the spraying is carried out for 40min, and the eye conjunctiva stent loaded with active particles is obtained so as to repair conjunctiva injury, and the volume ratio of the solution B to the solution C is 1:0.0.

And (5) after the electrostatic spinning is finished, placing the bracket in a fume hood at room temperature for 3 days to fully volatilize residual solvent, and then cutting the conjunctival bracket to any size to match the damaged conjunctival region.

Example 2

Step (1), dissolving polycaprolactone and gelatin (the mass ratio is 1:1) in trifluoroethanol, and magnetically stirring at room temperature for 12 hours to obtain a solution A with the mass concentration of 12%;

Step (2), carrying out electrostatic spinning by using the solution A, wherein when a receiver of the electrostatic spinning is a roller with the rotating speed of 1200rpm, the electrostatic spinning is deposited, the flowing rate of the solution A is 2ml/h, the voltage is 15Kv, the receiving distance is 15cm, the spinning is carried out for 40min, the rotating speed is reduced to 100rpm, and other spinning conditions are simultaneously carried out for 50min, so as to obtain the polycaprolactone-gelatin fiber film with the single-shaft ordered arrangement structure;

step (3), dissolving gelatin in acetic acid aqueous solution, magnetically stirring at room temperature for 12 hours, fully mixing to obtain 30mg/mL solution B, loading vascular endothelial growth factor, and dissolving in physiological saline to obtain 2mg/mL solution C;

And (4) coaxially and electrostatically spraying the mixture onto the polycaprolactone-gelatin fiber with the outer-layer single-axis ordered structure obtained in the step (2), wherein the dosage ratio of the fiber membrane with the single-axis ordered structure to the total volume of the solution B and the solution C is 1g of the fiber membrane with the single-axis ordered structure, 5mL of the solution is adopted, the advancing rate of the shell electrospray solution is 1.6mL/h, the advancing rate of the core electrospray solution is 0.4mL/h, the voltage is 20kV, the receiving distance is 15cm, and the spraying is carried out for 15min, so that the eye conjunctiva stent loaded with active particles is obtained to repair conjunctival injury, and the volume ratio of the shell solution to the core solution is 1:0.25.

And (5) after the electrostatic spinning is finished, placing the spinning film in a fume hood at room temperature for 3 days to fully volatilize residual solvents, and then cutting the conjunctival support to any size to match the damaged conjunctival region.

Example 3

Step (1), dissolving polylactic acid in chloroform, and magnetically stirring at room temperature for 24 hours to obtain a solution A with the mass concentration of 8%;

Step (2), carrying out electrostatic spinning by using a solution A, wherein when a receiver of the electrostatic spinning is a roller with the rotating speed of 1500rpm, the electrostatic spinning is deposited, the flowing rate of the solution A is 1.5ml/h, the voltage is 18Kv, the receiving distance is 20cm, the spinning is carried out for 60min, the rotating speed is reduced to 10rpm, and other spinning conditions are simultaneously carried out, and the polylactic acid fiber membrane with a single-shaft ordered arrangement structure is obtained;

Step (3), dissolving collagen in acetic acid aqueous solution, magnetically stirring at room temperature for 24 hours, fully mixing to obtain 40mg/mL shell solution B, loading epidermal growth factor, and dissolving in deionized water to obtain 50 mug/mL solution C;

Mixing the solutions B and C (volume ratio is 1:3), spraying uniaxially static electricity on the outer layer fiber with uniaxially ordered structure obtained in the step (2), wherein the dosage ratio of the fiber membrane with uniaxially ordered structure to the total volume of the solutions B and C is 1g of the fiber membrane with uniaxially ordered structure, 18mL of the solution, 25kV of the voltage, 3mL/h of the flow rate and 20cm of the receiving distance, and spraying for 15min to obtain the eye conjunctiva stent loaded with active particles so as to repair conjunctiva injury;

Example 4

Step (1), dissolving polycaprolactone and chitosan (the mass ratio is 0.2:1) in 70% acetic acid aqueous solution, and magnetically stirring at room temperature for 12 hours to obtain solution A with the mass concentration of 10%;

Step (2), carrying out electrostatic spinning by using the solution A, wherein when a receiver of the electrostatic spinning is a roller with the rotating speed of 1300rpm, the advancing speed of the solution A is 1.0ml/h, the voltage is 20kV, the receiving distance is 20cm, the spinning is carried out for 50min, the rotating speed is reduced to 50rpm, and other spinning conditions are adopted, and meanwhile, the spinning is carried out for 40 mm, so that the polycaprolactone-chitosan fiber membrane with the single-axis ordered arrangement structure is obtained;

mixing the solutions B and C (volume ratio of 1:0.4), and spraying uniaxially static electricity to the fiber with the outer layer uniaxially ordered structure obtained in the step (2), wherein the dosage ratio of the fiber film with the uniaxially ordered structure to the total volume of the solutions B and C is 1g of the fiber film with the uniaxially ordered structure, namely 5mL of the solution, 17kV of the voltage, 0.5mL/h of the flow rate, 20cm of the receiving distance and 50min of spraying to obtain an eye conjunctiva stent loaded with active particles so as to repair conjunctiva injury;

Example 5

Step (1), dissolving polycaprolactone and gelatin (the mass ratio is 10:1) in trifluoroethanol, and magnetically stirring at room temperature for 12 hours to obtain a solution A with the mass concentration of 10%;

Step (2), carrying out electrostatic spinning by using the solution A, wherein when a receiver of the electrostatic spinning is a roller with the rotating speed of 1300rpm, the advancing speed of the solution A is 1.2ml/h, the voltage is 25kV, the receiving distance is 20cm, the spinning is carried out for 50min, the rotating speed is reduced to 10rpm, and other spinning conditions are adopted, and meanwhile, the spinning is carried out for 40 mm, so that the polycaprolactone-gelatin fiber film with the single-axis ordered arrangement structure is obtained;

Step (3), dissolving collagen in an acetic acid aqueous solution, magnetically stirring at room temperature for 12 hours, fully mixing to obtain a 20mg/mL shell solution B, loading acidic fibroblast growth factor into physiological saline, and obtaining a 1mg/mL nuclear layer solution C;

mixing the solutions B and C (volume ratio is 1:1.2), and spraying uniaxially static electricity to the fiber with the outer layer uniaxially ordered structure obtained in the step (2), wherein the dosage ratio of the fiber film with the uniaxially ordered structure to the total volume of the solutions B and C is 1g of the fiber film with the uniaxially ordered structure, namely 10mL of the solution, 20kV of the voltage, the flow rate is 1.5mL/h, the receiving distance is 20cm, and spraying is carried out for 30min, so that the eye conjunctiva stent loaded with active particles is obtained to repair conjunctiva injury;

Fig. 1 is a schematic view of an conjunctival stent, which is a two-layer structure, 1 is an outer layer of uniaxially ordered fibers, and 2 is an inner layer of electrostatically sprayed active particles.

FIG. 2 is an SEM image of the polycaprolactone fiber obtained in step (2) of example 1, and it can be seen from the image that the fibers are arranged in uniaxial order.

Fig. 3 shows the results of cell proliferation experiments of the conjunctival stent prepared in example 1, and it can be seen from the figures that the absorbance of the blank group of the tissue culture dish and the absorbance of the conjunctival stent group 1,3 and 5 days are gradually increased, which indicates that the cells proliferate in the tissue culture dish and the conjunctival stent, and the absorbance of the conjunctival stent group 1,3 and 5 days is higher than that of the tissue culture dish, which proves that the conjunctival stent is safe and nontoxic and can promote cell proliferation and growth.

Fig. 4 is an SEM image of the conjunctival stent obtained in example 2, from which it can be seen that the fibers are uniaxially ordered and the electrostatically sprayed particles loaded with the active particles are uniformly distributed on the surface of the fibers.

Fig. 5 is a migration chart of fibroblasts in the conjunctival ocular stent obtained in example 3, from which it can be seen that the cells migrate directionally in the fibrous direction.

Fig. 6 is an SEM image of the polycaprolactone-chitosan fiber obtained in step (2) of example 1 of example 4, from which it can be seen that the fibers are arranged in uniaxial order.

Fig. 7 is an SEM image of the conjunctival stent obtained in example 5, from which it can be seen that the fibers are uniaxially ordered and the electrostatically sprayed particles loaded with the active particles are uniformly distributed on the surface of the fibers.

The conjunctival stent prepared in examples 1 to 5, wherein the electrostatic spinning fiber membrane is a fiber membrane with a uniaxial ordered structure, cells can be recruited through a surface contact induction effect, and the natural high molecular polymer in the active particles is beneficial to protecting the growth factors, meanwhile, a specific coarse structure or a patterning structure is constructed on the surface of the conjunctival stent, so that migration of the cells can be induced, the loaded growth factors can promote cell growth, and the repair effect of conjunctival injury is improved.

Claims

1. A method of preparing an conjunctival stent, the method comprising:

(4) Taking the fiber membrane with the uniaxial ordered arrangement structure obtained in the step (2) as a receiver, respectively taking the solution B and the solution C as a shell layer and a core layer for coaxial electrostatic spraying, or mixing the solution B and the solution C for uniaxial electrostatic spraying to obtain the conjunctival stent;

The preparation method comprises the following steps of (1), wherein the high molecular polymer is a mixture of a natural high molecular polymer B and a degradable synthetic high molecular polymer or the degradable synthetic high molecular polymer, and the degradable synthetic high molecular polymer is at least one of polylactic acid, polycaprolactone, a polylactic acid-glycolic acid copolymer and a polylactic acid-glycolic acid-caprolactone copolymer;

The natural high polymer A is at least one of collagen, gelatin, chitosan, starch, cellulose and elastin, and the growth factor is at least one of an epidermal growth factor, a nerve growth factor, a vascular endothelial growth factor, a platelet derived growth factor and an acidic fibroblast growth factor.

2. A method of preparing an eye conjunctiva stent according to claim 1, wherein:

step (1),

3. A method of preparing an eye conjunctiva stent according to claim 2, wherein:

The mass ratio of the degradable synthetic high molecular polymer to the natural high molecular polymer in the mixture of the natural high molecular polymer B and the degradable synthetic high molecular polymer is less than 100, and/or,

The mass concentration of the solution A is 5-24%.

4. A method of preparing an eye conjunctiva stent according to claim 3, wherein:

the mass ratio of the degradable synthetic high molecular polymer to the natural high molecular polymer in the mixture of the natural high molecular polymer B and the degradable synthetic high molecular polymer is (0.1-10): 1, and/or,

The mass concentration of the solution A is 6-12%.

5. A method of preparing an eye conjunctiva stent according to claim 1, wherein:

Step (2),

The pushing rate of the solution A is 0.1-10 mL/h, and/or,

The voltage is 8-30 kV, and/or,

The receiving distance is 10-25 cm, and/or,

The initial drum rotation speed is 800-3000 rpm, the spinning time is 10-480 min, and/or,

Reducing the rotating speed of the roller to 10-200 rpm; the spinning time is 10-480 min.

6. A method of preparing an eye conjunctiva stent according to claim 5, wherein:

Step (2),

The pushing rate of the solution A is 1-2 mL/h, and/or,

The voltage is 15-25 kV, and/or,

The receiving distance is 15-20 cm, and/or,

The rotation speed of the initial roller is 1000-2000 rpm, the spinning time is 30-120 min, and/or,

Reducing the rotating speed of the roller to 10-100 rpm; the spinning time is 30-120 min.

7. A method of preparing an eye conjunctiva stent according to claim 6, wherein:

Step (2),

The rotation speed of the initial roller is 1000-1500 rpm, the spinning time is 40-60 min, and/or,

The rotating speed of the roller is reduced to 10-100 rpm, the spinning time is 40-60 min.

8. A method of preparing an eye conjunctiva stent according to claim 1, wherein:

Step (3),

The solvent B is at least one of hydrochloric acid, acetic acid, ethyl acetate, glycerol, trifluoroethanol, hexafluoroisopropanol and water, and/or,

The solvent C is at least one of normal saline, ultrapure water, deionized water, buffer solution, methanol and ethanol.

9. A method of preparing an eye conjunctiva stent according to claim 1, wherein:

Step (3),

The concentration of the solution B is 1 mg/mL-200 mg/mL;

the concentration of the solution C is 10 mug/mL-10 mg/mL.

10. A method of preparing an eye conjunctiva stent according to claim 9, wherein:

Step (3),

The concentration of the solution B is 10 mg/mL-50 mg/mL;

the concentration of the solution C is 50 mug/mL-5 mg/mL.

11. A method of preparing an eye conjunctiva stent according to claim 10, wherein:

Step (3),

The concentration of the solution B is 20 mg/mL-40 mg/mL;

the concentration of the solution C is 50 mug/mL-2 mg/mL.

12. A method of preparing an eye conjunctiva stent according to claim 1, wherein:

Step (4),

The dosage ratio of the fiber film with the uniaxially ordered structure to the total volume of the solution B and the solution C is 1g of the fiber film with the uniaxially ordered structure (1-60 mL of the solution), and/or,

When coaxial electrostatic spraying is performed, the volume ratio of the solution B to the solution C is 1 (0.1-1), and/or,

In the case of uniaxial electrostatic spraying, the volume ratio of the solution B to the solution C is 1 (0.1-10).

13. A method of preparing an eye conjunctiva stent according to claim 12, wherein:

Step (4),

The dosage ratio of the fiber film with the uniaxially ordered structure to the total volume of the solution B and the solution C is 1g of the fiber film with the uniaxially ordered structure (5-20 mL of the solution), and/or,

When coaxial electrostatic spraying is performed, the volume ratio of the solution B to the solution C is 1 (0.2-0.4), and/or,

In the case of uniaxial electrostatic spraying, the volume ratio of the solution B to the solution C is 1 (0.3-3).

14. A method of preparing an eye conjunctiva stent according to claim 1, wherein:

Step (4),

In the coaxial electrostatic spraying process, the propelling speed of the solution B electrospraying solution is 0.1-10 mL/h, the propelling speed of the solution C electrospraying solution is 0.1-5 mL/h, the voltage is 8-30 kV, the receiving distance is 8-30 cm, and the spraying time is 5-360 min;

In the single-axis electrostatic spraying process, the advancing rate of the electrospray solution after the solution B and the solution C are mixed is 0.5-10 mL/h, the voltage is 8-30 kV, the receiving distance is 8-30 cm, and the spraying time is 5-360 min.

15. A method of preparing an eye conjunctiva stent according to claim 14, wherein:

Step (4),

In the coaxial electrostatic spraying process, the pushing rate of the solution B electrospraying solution is 1-3 mL/h, the pushing rate of the solution C electrospraying solution is 0.4-1 mL/h, the voltage is 15-25 kV, the receiving distance is 15-20 cm, and the spraying time is 15-120 min;

In the single-axis electrostatic spraying process, the advancing rate of the electrospray solution after the solution B and the solution C are mixed is 0.5-3 mL/h, the voltage is 15-25V, the receiving distance is 10-25 cm, and the spraying time is 15-120 min.

16. A method of preparing an eye conjunctiva stent according to claim 15, wherein:

Step (4),

In the coaxial electrostatic spraying process, the spraying time is 15-40 min;

And in the case of uniaxial electrostatic spraying, the spraying time is 15-60 min.

17. An conjunctival stent prepared by the method of any one of claims 1 to 16, wherein:

The conjunctiva support is of a two-layer structure;

the outer layer is an electrostatic spinning fiber with a single-axis ordered arrangement structure;

the inner layer is an electrostatically sprayed layer loaded with active particles.

18. Use of an ocular conjunctival stent prepared by the method of any one of claims 1 to 16 for repairing conjunctival lesions.