Preparation method of conjunctival stent, conjunctival stent and application thereof

Abstract

The present invention provides a preparation method of a conjunctival scaffold, a conjunctival scaffold and an application thereof. The method comprises: adding a high molecular polymer to a solvent A, dissolving to obtain a solution A; using a roller as a receiver to electrostatically spin the solution A to obtain a fiber membrane with a uniaxial ordered arrangement structure; adding a natural high molecular polymer A to a solvent B, dissolving to obtain a solution B; adding a growth factor to a solvent C, dissolving to obtain a solution C; using a fiber membrane with a uniaxial ordered arrangement structure as a receiver, using solution B and solution C as a shell layer and a core layer respectively for coaxial electrostatic spraying, or mixing solution B and solution C for uniaxial electrostatic spraying to obtain the conjunctival scaffold. The conjunctival scaffold of the present invention has the function of inducing directional migration of epithelial cells and promoting cell growth, can accelerate conjunctival regeneration, and the conjunctival scaffold can be degraded and replaced by a new conjunctiva, or degraded and separated from the conjunctival surface after healing.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, and more particularly to a preparation method of a conjunctival stent, a conjunctival stent and applications thereof. Background Art

[0002] The conjunctiva is a layer of translucent mucosal tissue that starts from the end of the inter-eyelid margin and covers the surface of the sclera behind the eyelid and the anterior segment of the eyeball. When the conjunctiva is damaged by various traumas, thermal burns, chemical burns or severe ocular surface diseases, it will cause defects in conjunctival tissue. Autologous conjunctival transplantation, allogeneic conjunctival transplantation or amniotic membrane transplantation are now commonly used in clinical practice to repair conjunctival defects. The normal conjunctiva that can be used for autologous transplantation is limited and is a method of "taking money from one pocket to fill another", which will cause damage to itself. The normal conjunctiva that can be used for allogeneic transplantation also faces the problem of material shortage and the risk of immune rejection. Amniotic membrane transplantation also has its shortcomings. In the inflammatory environment of the eye, the amniotic membrane will degrade rapidly and cannot effectively guarantee the growth of epithelial cells for a long time. In addition, individual differences in amniotic membrane donors make the thickness and quality of amniotic membrane transplants different, and there is also a risk of infectious disease infection.

[0003] Therefore, in order to solve the problem of insufficient donors for autologous and allogeneic conjunctival transplantation and the rapid degradation rate of amniotic membrane transplantation, it is of great significance to develop an artificial conjunctival scaffold with an appropriate degradation time and the ability to accelerate the repair of conjunctival defects. Summary of the invention

[0004] In order to solve the technical problems existing in the prior art, the present invention provides a preparation method of a conjunctival stent, a conjunctival stent and applications.

[0005] The artificial conjunctiva stent of the present invention has a suitable degradation time and can accelerate the repair of conjunctiva defects. After the human conjunctiva is damaged, the artificial conjunctiva stent can be sutured to the damaged part to accelerate the repair of the conjunctiva and degrade after the conjunctiva is repaired.

[0006] The present invention constructs an electrostatically spun fiber with a uniaxial orderly arrangement structure as an outer layer, and the combined active particles as an inner layer (the inner layer is an electrostatic spray layer loaded with a core-shell structure of active particles, the core layer contains growth factors, and the shell layer is a natural high molecular polymer), etc., to continuously release the required active particles from the conjunctival scaffold; the outer layer is an electrostatically spun fiber with a uniaxial orderly arrangement structure, which guides the directional migration and proliferation of epithelial cells; the active particles containing growth factors promote the growth of epithelial cells, and the conjunctival scaffold can be degraded and replaced by new conjunctiva, or degraded and separated on the surface of the conjunctiva after healing.

[0007] The conjunctival stent of the present invention 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 to improve the biocompatibility of the conjunctival stent, control the degradation rate, and load active particles to promote the repair rate of conjunctival damage.

[0008] One of the purposes of the present invention is to provide a method for preparing a conjunctival stent, comprising:

[0009] (1) adding a high molecular weight polymer into a solvent A and dissolving the polymer to obtain a solution A;

[0010] (2) electrospinning the solution A obtained in step (1) using a roller as a receiver to obtain a fiber membrane with a uniaxial ordered arrangement structure;

[0011] (3) adding natural polymer A to solvent B and dissolving it to obtain solution B; adding growth factor to solvent C and dissolving it to obtain solution C;

[0012] (4) Using the fiber membrane with a uniaxial ordered structure obtained in step (2) as a receiver, solution B and solution C are used as the shell layer and core layer respectively for coaxial electrostatic spraying, or solution B and solution C are mixed for uniaxial electrostatic spraying to obtain the conjunctival scaffold.

[0013] In a preferred embodiment of the present invention,

[0014] Step (1),

[0015] The high molecular polymer is a mixture of a natural high molecular polymer B and a degradable synthetic high molecular polymer, or a degradable synthetic high molecular polymer; the degradable synthetic high molecular polymer is preferably at least one of polylactic acid, polycaprolactone, polylactic acid-glycolic acid copolymer, polylactic acid-glycolic acid-caprolactone copolymer; the natural high molecular polymer B is preferably at least one of collagen, gelatin, chitosan, starch, cellulose, and elastin;

[0016] The solvent A is at least one of hexafluoroisopropanol, trifluoroethanol, chloroform, methanol, dichloromethane, acetic acid and its aqueous solution, and N,N'-dimethylformamide.

[0017] In a preferred embodiment of the present invention,

[0018] The mass ratio of the degradable synthetic polymer to the natural polymer in the mixture of the natural polymer B and the degradable synthetic polymer is less than 100, and the mass ratio is preferably (0.1-10):1;

[0019] The mass concentration of the solution A is 5 to 24%, preferably 6 to 12%.

[0020] In a preferred embodiment of the present invention,

[0021] Step (2),

[0022] The advancement rate of solution A is 0.1 to 10 mL / h, preferably 1 to 2 mL / h;

[0023] The voltage is 8 to 30 kV, preferably 15 to 25 kV;

[0024] The receiving distance is 10 to 25 cm, preferably 15 to 20 cm;

[0025] The roller speed is first high speed and then low speed:

[0026] The initial roller speed is 800 to 3000 rpm, preferably 1000 to 2000 rpm, more preferably 1000 to 1500 rpm; the spinning time at this high speed is 10 to 480 min, preferably 30 to 120 min, more preferably 40 to 60 min;

[0027] Then the roller speed is reduced to 10-200 rpm, preferably 10-100 rpm; the spinning time at this low speed is 10-480 min, preferably 30-120 min, more preferably 40-60 min.

[0028] By collecting electrospun fibers through a high-speed rotating (greater than 800rpm) roller collector, uniaxially ordered fibers can be obtained. The uniaxial ordered arrangement is conducive to the directional migration and growth of cells and is expected to accelerate the repair of the conjunctiva. By collecting through a low-speed rotating (less than 200rpm) roller collector, randomly arranged fibers can be obtained, providing mechanical strength to the material.

[0029] In a preferred embodiment of the present invention,

[0030] Step (3),

[0031] The natural high molecular polymer A is at least one of collagen, gelatin, chitosan, starch, cellulose and elastin;

[0032] The solvent B is at least one of hydrochloric acid, acetic acid, ethyl acetate, glycerol, trifluoroethanol, and hexafluoroisopropanol;

[0033] The solvent C is at least one of physiological saline, ultrapure water, buffer solution, methanol, and ethanol;

[0034] The growth factor is at least one of epidermal growth factor, nerve growth factor, vascular endothelial growth factor, platelet-derived growth factor and acidic fibroblast growth factor.

[0035] In a preferred embodiment of the present invention,

[0036] Step (3),

[0037] The concentration of solution B is 1 mg / mL to 200 mg / mL; preferably 10 mg / mL to 50 mg / mL, more preferably 20 mg / mL to 40 mg / mL;

[0038] The concentration of solution C is 10 μg / mL to 10 mg / mL, preferably 50 μg / mL to 5 mg / mL, and more preferably 50 μg / mL to 2 mg / mL.

[0039] In a preferred embodiment of the present invention,

[0040] Step (4),

[0041] The ratio of the uniaxially ordered fiber membrane to the total volume of solution B and solution C is 1 g of the uniaxially ordered fiber membrane: (1-60) mL of solution, preferably 1 g of the uniaxially ordered fiber membrane: (5-20) mL of solution;

[0042] During coaxial electrostatic spraying, the volume ratio of solution B to solution C is 1:(0.1-1); preferably 1:(0.2-0.4);

[0043] During uniaxial electrostatic spraying, the volume ratio of solution B to solution C is 1:(0.1-10), preferably 1:(0.3-3).

[0044] In a preferred embodiment of the present invention,

[0045] Step (4),

[0046] During coaxial electrostatic spraying, the electrospraying solution propulsion rate of solution B is 0.1-10 mL / h, preferably 1-3 mL / h; the electrospraying solution propulsion rate of solution C 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; the spraying time is 5-360 min, preferably 15-120 min, more preferably 15-40 min;

[0047] During uniaxial electrostatic spraying, the propulsion rate of the electrosprayed solution after the mixture of solution B and solution C is 0.5-10 mL / h, preferably 0.5-3 mL / h; the voltage is 8-30 kV, preferably 15-25 V; the receiving distance is 8-30 cm, preferably 10-25 cm; the spraying time is 5-360 min, preferably 15-120 min, and more preferably 15-60 min.

[0048] The amount of nanoparticles deposited on the fiber membrane is adjusted by adjusting the spray time to match the solution concentration and propulsion speed; during coaxial electrostatic spraying, solution B will wrap solution C, which will have a sustained release effect on the growth factor, and the uniaxial method is a mixture of the two substances, which will release the growth factor faster; when the damage is large, the growth factor needs to be released slowly, and the conjunctival stent made by coaxial electrostatic spraying can be used; when the damage is small, the factor needs to be released quickly, and the conjunctival stent made by uniaxial electrostatic spraying can be used.

[0049] A second object of the present invention is to provide a conjunctival stent.

[0050] The conjunctival stent is a two-layer structure; the outer layer is electrostatic spinning fibers with a uniaxial orderly arrangement structure; and the inner layer is an electrostatic spray layer loaded with active particles.

[0051] The conjunctival stent can be cut into any size according to actual needs. According to general clinical needs, the preferred length range is 3 mm to 30 mm and the width range is 2 mm to 20 mm.

[0052] The third purpose of the present invention is to provide an application of a conjunctival stent in repairing conjunctival damage.

[0053] The present invention can specifically adopt the following technical solutions:

[0054] The preparation method of the present invention specifically comprises the following steps:

[0055] 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 them to obtain a solution A;

[0056] Step (2), electrospinning solution A using a high-speed rotating roller as a receiver to obtain a fiber membrane with a uniaxial ordered arrangement structure;

[0057] Step (3), adding the natural high molecular polymer to solvent B to obtain solution B; adding the growth factor to solvent C to obtain solution C;

[0058] Step (4), using the uniaxial ordered fiber membrane obtained in step (2) as a receiver, and using solution B and solution C as the shell layer and core layer respectively for coaxial electrostatic spraying, or mixing solution B and solution C for uniaxial electrostatic spraying, to obtain a uniaxial ordered structure fiber membrane loaded with active particles; and obtaining the conjunctival scaffold loaded with active particles.

[0059] The nanofiber membrane obtained by the electrostatic spraying needs to be placed in a fume hood for 2 to 3 days to remove the solvent.

[0060] Compared with the prior art, the present invention has the following beneficial effects:

[0061] The artificially synthesized conjunctival stent provided by the present invention plays a role in repairing the membrane, solving the problem of insufficient donors for autologous or allogeneic conjunctival transplantation, and at the same time using a polymer with adjustable degradation rate to solve the problem of too fast degradation during amniotic membrane repair.

[0062] The conjunctival stent of the present invention is prepared by electrostatic spraying. During spraying, nanoparticles are sprayed onto fibers in the form of droplets. The droplets adhere to the fibers. The nanoparticles formed after drying have stronger binding force with the fibers and only exist on the side facing the conjunctival damage. The particle loading amount can be changed by adjusting the concentrations of solution B and solution C and related parameters of electrostatic spraying.

[0063] The conjunctival scaffold prepared by the present invention can obtain uniaxial orderly arranged fibers by collecting electrospun fibers through a roller collector rotating at a high speed (greater than 800rpm), and this side contacts the conjunctival wound. Compared with the random electrospun fiber membrane, the ordered uniaxial orderly arranged fibers can recruit cells through the surface contact induction effect, and the natural high molecular polymer in the active particles is conducive to protecting the growth factor. At the same time, a specific rough structure or patterned structure is constructed on the surface of the conjunctival scaffold, which can induce cell migration. The loaded growth factor can promote cell growth and improve the repair effect of conjunctival damage. And the randomly arranged fibers can be obtained by collecting with a roller collector rotating at a low speed (less than 200rpm), and this side does not directly contact the conjunctival wound, providing better mechanical strength for the membrane material.

[0064] The conjunctival stent provided by the present invention can be cut into different sizes to match the size of the damaged conjunctiva, thereby improving the repair effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 This is a schematic diagram of the conjunctival stent;

[0066] 1 is uniaxial ordered fibers, 2 is electrostatic spray particles;

[0067] Figure 2 This is a SEM image of the polycaprolactone uniaxially ordered fibers obtained in step (2) of Example 1;

[0068] Figure 3 This is a diagram showing the results of a cell proliferation experiment on the conjunctival scaffold prepared in Example 1;

[0069] Figure 4 This is a SEM image of the conjunctival scaffold obtained in Example 2;

[0070] Figure 5 This is a migration diagram of fibroblasts in the conjunctival scaffold obtained in Example 3;

[0071] Figure 6This is a SEM image of the polycaprolactone-chitosan fiber obtained in step (2) of Example 1 of Example 4;

[0072] Figure 7 This is the SEM image of the conjunctiva scaffold obtained in Example 5. DETAILED DESCRIPTION

[0073] The present invention is described in detail below in conjunction with specific drawings and embodiments. It is necessary to point out that the following embodiments are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made to the present invention by those skilled in the art based on the content of the present invention still fall within the scope of protection of the present invention.

[0074] The raw materials used in the examples are all conventional commercially available raw materials.

[0075] Test method:

[0076] The proliferation of cells was detected by CCK-8 method. The materials that had been disinfected and sterilized were cut into materials with a diameter of 1.5 cm and spread on the bottom of a 24-well plate. L929 that had grown to cover the bottom of the culture bottle was taken for seeding. The cell suspension was transferred to the well plate at 100 μL / well and seeded on the blank wells and the surface of the material. Then 900 μL of cell culture fluid was added to each well, and the 24-well plate after seeding was placed in a cell culture incubator. CCK-8 test was performed on 1 day, 3 days, and 5 days. After the original culture fluid was removed from each well, a CCK-8 solution obtained by mixing the cell culture fluid and 10% of its volume of CCK-8 reagent was prepared and added to it at 500 μL / well. After the well plate was placed in the cell culture incubator for 4 hours, its absorbance value at a wavelength of 450nm was tested.

[0077] Fibroblast migration: Before fibroblast inoculation, the conjunctival scaffold was washed three times with cell culture medium. Then, a sterilized polydimethylsiloxane strip was placed on the sample, leaving an area with a width of 0.5 cm on the left side to inoculate fibroblasts. After 4 hours of cell adhesion, the strip was removed to allow the cells to migrate forward. After another three days of incubation, the cells were stained separately. The cells were fixed in a 3% glutaraldehyde solution for 10 minutes, permeabilized with 0.1% Triton X-100 for 5 minutes, and then blocked with a 3% BSA solution for 1 hour at room temperature. Then they were immersed in solutions of Alexa Fluor 555 phalloidin and 4',6-diamidino-2-phenylindole for 20 and 5 minutes at room temperature, respectively. Each operation was washed three times with PBS solution. After staining, the samples were observed and photographed under a laser confocal scanning microscope.

[0078] Example 1

[0079] Step (1) polycaprolactone was dissolved in dichloromethane and magnetically stirred at room temperature for 12 h to obtain a solution A having a mass concentration of 10%;

[0080] Step (2) electrospinning with solution A, the receiver of electrospinning was a roller with a rotation speed of 1000 rpm, the advancement rate of solution A was 1 ml / h, the voltage was 15 kV, the receiving distance was 20 cm, spinning for 60 min, reducing the speed to 10 rpm, other spinning conditions remained unchanged, spinning for 40 min, to obtain a uniaxial ordered structure of polycaprolactone fiber membrane;

[0081] Step (3) Dissolve collagen in aqueous acetic acid solution, stir magnetically at room temperature for 12 h and mix thoroughly to obtain a 20 mg / mL shell solution B, load epidermal growth factor and dissolve it in saline to obtain a 1 mg / mL core solution C;

[0082] Step (4). Coaxial electrostatic spraying is performed on the outer layer of the uniaxially ordered structured polycaprolactone fiber obtained in step (2). The ratio of the amount of the uniaxially ordered structured fiber membrane to the total volume of solution B and solution C is 1g of uniaxially ordered structured fiber membrane: 18mL of solution. The shell layer electrospraying solution propulsion rate is 3mL / h, the core layer electrospraying solution propulsion rate is 1mL / h, the voltage is 22kV, the receiving distance is 18cm, and the spraying is performed for 40min to obtain an eye conjunctival scaffold loaded with active particles to repair conjunctival damage. The volume ratio of solution B to solution C is 1:0.33.

[0083] Step (5). After the electrospinning is completed, the scaffold is placed in a fume hood at room temperature for 3 days to allow the residual solvent to fully evaporate. The conjunctival scaffold can then be cut into any size to match the damaged area of ​​the conjunctiva.

[0084] Example 2

[0085] Step (1). Dissolve polycaprolactone and gelatin (mass ratio 1:1) in trifluoroethanol and stir magnetically at room temperature for 12 h to obtain a solution A with a mass concentration of 12%;

[0086] Step (2). Electrospinning was performed using solution A. When the electrospinning receiver was a roller with a rotation speed of 1200 rpm, electrospinning deposition was performed. The flow rate of solution A was 2 ml / h, the voltage was 15 Kv, the receiving distance was 15 cm, the spinning time was 40 min, the speed was reduced to 100 rpm, and the other spinning conditions were maintained at the same time. The spinning time was 50 min to obtain a polycaprolactone-gelatin fiber membrane with a uniaxial ordered arrangement structure;

[0087] Step (3) gelatin was dissolved in an aqueous acetic acid solution, magnetically stirred at room temperature for 12 h and thoroughly mixed to obtain a 30 mg / mL solution B, which was loaded with vascular endothelial growth factor dissolved in saline to obtain a 2 mg / mL solution C;

[0088] Step (4). Coaxial electrostatic spraying is performed on the outer layer of the uniaxially ordered structured polycaprolactone-gelatin fiber obtained in step (2). The ratio of the total volume of the fiber membrane with the uniaxially ordered structure to the solution B and the solution C is 1g of the fiber membrane with the uniaxially ordered structure: 5mL of the solution. The propulsion rate of the shell layer electrospraying solution is 1.6mL / h, the propulsion rate of the core layer electrospraying solution is 0.4mL / h, the voltage is 20kV, the receiving distance is 15cm, and the spraying is performed for 15min to obtain an eye conjunctival scaffold loaded with active particles to repair conjunctival damage. The volume ratio of the shell layer solution to the core layer solution is 1:0.25.

[0089] Step (5). After the electrospinning is completed, the spun membrane is placed in a fume hood at room temperature for 3 days to allow the residual solvent to fully evaporate. The conjunctival scaffold can then be cut into any size to match the damaged area of ​​the conjunctiva.

[0090] Example 3

[0091] Step (1) polylactic acid was dissolved in chloroform and magnetically stirred at room temperature for 24 hours to obtain a solution A having a mass concentration of 8%;

[0092] Step (2) electrospinning with solution A, the receiver of electrospinning was a roller with a rotation speed of 1500 rpm, electrospinning deposition, solution A flow rate of 1.5 ml / h, voltage 18Kv, receiving distance of 20 cm, spinning for 60min, reducing the speed to 10rpm, while other spinning conditions, spinning for 60min, to obtain a uniaxial ordered structure of polylactic acid fiber membrane;

[0093] Step (3) Dissolve collagen in aqueous acetic acid solution, stir magnetically at room temperature for 24 h and mix thoroughly to obtain a 40 mg / mL shell solution B, load epidermal growth factor and dissolve it in deionized water to obtain a 50 μg / mL solution C;

[0094] Step (4). Mix solutions B and C (volume ratio 1:3), and use uniaxial electrostatic spraying to the outer layer of uniaxial ordered structure fibers obtained in step (2), the dosage ratio of the uniaxial ordered structure fiber membrane to the total volume of solution B and solution C is 1g uniaxial ordered structure fiber membrane: 18mL solution, voltage 25kV, flow rate 3ml / h, receiving distance 20cm, spraying 15min, to obtain an eye conjunctival scaffold loaded with active particles to repair conjunctival damage;

[0095] Step (5). After the electrospinning is completed, the spun membrane is placed in a fume hood at room temperature for 3 days to allow the residual solvent to fully evaporate. The conjunctival scaffold can then be cut into any size to match the damaged area of ​​the conjunctiva.

[0096] Example 4

[0097] Step (1) polycaprolactone and chitosan (mass ratio 0.2:1) were dissolved in 70% acetic acid aqueous solution and magnetically stirred at room temperature for 12 hours to obtain a solution A with a mass concentration of 10%;

[0098] Step (2) electrospinning with solution A, the receiver of electrospinning was a roller with a rotation speed of 1300 rpm, the advancement rate of solution A was 1.0 ml / h, the voltage was 20 kV, the receiving distance was 20 cm, spinning for 50 min, reducing the speed to 50 rpm, and spinning for 40 min under other spinning conditions to obtain a uniaxial ordered structure of polycaprolactone - chitosan fiber membrane;

[0099] Step (3) Dissolve collagen in aqueous acetic acid solution, stir magnetically at room temperature for 12 h and mix thoroughly to obtain a 20 mg / mL shell solution B, load epidermal growth factor and dissolve it in saline to obtain a 1 mg / mL core solution C;

[0100] Step (4). Mix solutions B and C (volume ratio 1:0.4), and use uniaxial electrostatic spraying to the outer layer of uniaxial ordered structure fiber obtained in step (2), the dosage ratio of uniaxial ordered structure fiber membrane to the total volume of solution B and solution C is 1g uniaxial ordered structure fiber membrane: 5mL solution, voltage 17kV, flow rate 0.5ml / h, receiving distance 20cm, spraying 50min, to obtain an eye conjunctival scaffold loaded with active particles to repair conjunctival damage;

[0101] Step (5). After the electrospinning is completed, the scaffold is placed in a fume hood at room temperature for 3 days to allow the residual solvent to fully evaporate. The conjunctival scaffold can then be cut into any size to match the damaged area of ​​the conjunctiva.

[0102] Example 5

[0103] Step (1). Dissolve polycaprolactone and gelatin (mass ratio 10:1) in trifluoroethanol and stir magnetically at room temperature for 12 h to obtain a solution A with a mass concentration of 10%;

[0104] Step (2) electrospinning with solution A, the receiver of electrospinning was a roller with a rotation speed of 1300 rpm, the propulsion rate of solution A was 1.2 ml / h, the voltage was 25 kV, the receiving distance was 20 cm, spinning for 50 min, the speed was reduced to 10 rpm, and the other spinning conditions were simultaneously spun for 40 min to obtain a polycaprolactone-gelatin fiber membrane with a uniaxial ordered arrangement structure;

[0105] Step (3) Dissolve collagen in aqueous acetic acid solution, stir magnetically at room temperature for 12 h and mix thoroughly to obtain a 20 mg / mL shell solution B, load acidic fibroblast growth factor and dissolve in saline to obtain a 1 mg / mL core solution C;

[0106] Step (4). Mix solutions B and C (volume ratio 1:1.2), and use uniaxial electrostatic spraying to the outer layer of uniaxial ordered structure fiber obtained in step (2), the dosage ratio of uniaxial ordered structure fiber membrane to the total volume of solution B and solution C is 1g uniaxial ordered structure fiber membrane: 10mL solution, voltage 20kV, flow rate 1.5ml / h, receiving distance 20cm, spraying for 30min, to obtain an eye conjunctival scaffold loaded with active particles to repair conjunctival damage;

[0107] Step (5). After the electrospinning is completed, the scaffold is placed in a fume hood at room temperature for 3 days to allow the residual solvent to fully evaporate. The conjunctival scaffold can then be cut into any size to match the damaged area of ​​the conjunctiva.

[0108] Figure 1 This is a schematic diagram of the conjunctival stent. The conjunctival stent has a two-layer structure. 1 is the outer layer of uniaxial orderly arranged fibers, and 2 is the electrostatic spray inner layer loaded with active particles.

[0109] Figure 2 This is a SEM image of the polycaprolactone fibers obtained in step (2) of Example 1. It can be seen from the image that the fibers are arranged in a uniaxial order.

[0110] Figure 3 The results of the cell proliferation experiment of the conjunctival scaffold prepared in Example 1. It can be seen from the figure that the absorbance of the blank group of the tissue culture dish and the conjunctival scaffold group on days 1, 3, and 5 gradually increased, indicating that the cells proliferated in the tissue culture dish and the conjunctival scaffold. The absorbance of the conjunctival scaffold group on days 1, 3, and 5 was higher than that of the tissue culture dish, proving that the conjunctival scaffold is safe and non-toxic and can promote cell proliferation and growth.

[0111] Figure 4 This is a SEM image of the conjunctival scaffold obtained in Example 2. It can be seen from the image that the fibers are arranged in a uniaxial order, and the electrostatic spray particles loaded with active particles are evenly distributed on the fiber surface.

[0112] Figure 5 This is a migration diagram of fibroblasts in the conjunctival scaffold obtained in Example 3. It can be seen from the figure that the cells migrate directionally along the fiber direction.

[0113] Figure 6 This is a SEM image of the polycaprolactone-chitosan fibers obtained in step (2) of Example 1 of Example 4. It can be seen from the image that the fibers are uniaxially arranged in an orderly manner.

[0114] Figure 7 This is a SEM image of the conjunctival scaffold obtained in Example 5. It can be seen from the image that the fibers are arranged in a uniaxial order, and the electrostatic spray particles loaded with active particles are evenly distributed on the fiber surface.

[0115] The conjunctival scaffolds prepared in Examples 1 to 5 are electrospun fiber membranes with a uniaxial ordered arrangement structure, which can recruit cells through the surface contact induction effect. The natural high molecular polymers in the active particles are beneficial to protecting growth factors. At the same time, specific rough structures or patterned structures are constructed on the surface of the conjunctival scaffold, which can induce cell migration. The loaded growth factors can promote cell growth and improve the repair effect of conjunctival damage.

Claims

1. A method for preparing a conjunctival scaffold, characterized in that The method comprises: (1) Adding a high molecular weight polymer into solvent A and dissolving the polymer to obtain solution A; (2) electrospinning the solution A obtained in step (1) using a roller as a receiver to obtain a fiber membrane with a uniaxial ordered structure; (3) Adding natural polymer A to solvent B and dissolving it to obtain solution B; adding growth factor to solvent C and dissolving it to obtain solution C; (4) using the fiber membrane with a uniaxial ordered structure obtained in step (2) as a receiver, and using solution B and solution C as the shell layer and core layer respectively for coaxial electrostatic spraying, or mixing solution B and solution C for uniaxial electrostatic spraying, to obtain the conjunctival scaffold; Step (1), the polymer is a mixture of a natural polymer B and a degradable synthetic polymer, or a degradable synthetic polymer; the degradable synthetic polymer is at least one of polylactic acid, polycaprolactone, polylactic acid-glycolic acid copolymer, polylactic acid-glycolic acid-caprolactone copolymer; the natural polymer B is at least one of collagen, gelatin, chitosan, starch, cellulose, and elastin; In step (3), the natural high molecular polymer A is at least one of collagen, gelatin, chitosan, starch, cellulose, and elastin; and the growth factor is at least one of epidermal growth factor, nerve growth factor, vascular endothelial growth factor, platelet-derived growth factor, and acidic fibroblast growth factor.

2. The method for preparing the conjunctival scaffold according to claim 1, characterized in that: Step (1), The solvent A is at least one of hexafluoroisopropanol, trifluoroethanol, chloroform, methanol, dichloromethane, acetic acid and its aqueous solution, and N,N'-dimethylformamide.

3. The method for preparing the conjunctival scaffold according to claim 2, characterized in that: The mass ratio of the degradable synthetic polymer to the natural polymer in the mixture of the natural polymer B and the degradable synthetic polymer is less than 100; and / or, The mass concentration of the solution A is 5-24%.

4. The method for preparing the conjunctival scaffold according to claim 3, characterized in that: The mass ratio of the degradable synthetic polymer to the natural polymer in the mixture of the natural polymer B and the degradable synthetic polymer is (0.1-10):1; and / or, The mass concentration of the solution A is 6-12%.

5. The method for preparing the conjunctival scaffold according to claim 1, characterized in that: Step (2), Solution A advancing rate is 0.1~10mL / h; and / or, Voltage is 8~30kV; and / or, The receiving distance is 10~25cm; and / or, The initial roller speed is 800-3000 rpm; the spinning time is 10-480 min; and / or, Reduce the roller speed to 10~200 rpm; the spinning time is 10~480min.

6. The method for preparing the conjunctival scaffold according to claim 5, characterized in that: Step (2), Solution A advancing rate is 1~2mL / h; and / or, Voltage is 15~25kV; and / or, The receiving distance is 15~20cm; and / or, The initial roller speed is 1000-2000 rpm; the spinning time is 30-120 min; and / or, Reduce the roller speed to 10~100rpm; the spinning time is 30~120min.

7. The method for preparing the conjunctival scaffold according to claim 6, characterized in that: Step (2), The initial roller speed is 1000-1500 rpm; the spinning time is 40-60 min; and / or, Reduce the roller speed to 10~100rpm and the spinning time to 40~60min.

8. The method for preparing the conjunctival scaffold according to claim 1, characterized in that: Step (3), Solvent B is at least one of hydrochloric acid, acetic acid, ethyl acetate, glycerol, trifluoroethanol, hexafluoroisopropanol, and water; and / or, Solvent C is at least one of physiological saline, ultrapure water, deionized water, buffer solution, methanol, and ethanol.

9. The method for preparing the conjunctival scaffold according to claim 1, characterized in that: Step (3), The concentration of solution B is 1 mg / mL~200 mg / mL; The concentration of solution C is 10μg / mL~10mg / mL.

10. The method for preparing the conjunctival scaffold according to claim 9, characterized in that: Step (3), The concentration of solution B is 10 mg / mL~50 mg / mL; The concentration of solution C is 50μg / mL~5mg / mL.

11. The method for preparing the conjunctival scaffold according to claim 10, characterized in that: Step (3), The concentration of solution B is 20 mg / mL~40 mg / mL; The concentration of solution C is 50 μg / mL~2 mg / mL.

12. The method for preparing the conjunctival scaffold according to claim 1, characterized in that: Step (4), The dosage ratio of the fiber membrane with uniaxial ordered arrangement structure to the total volume of solution B and solution C is 1 g fiber membrane with uniaxial ordered arrangement structure: (1-60) mL solution; and / or, During coaxial electrostatic spraying, the volume ratio of solution B to solution C is 1:(0.1~1); and / or, During uniaxial electrostatic spraying, the volume ratio of solution B to solution C is 1:(0.1~10).

13. The method for preparing the conjunctival scaffold according to claim 12, characterized in that: Step (4), The ratio of the fiber membrane with uniaxial ordered arrangement structure to the total volume of solution B and solution C is 1 g fiber membrane with uniaxial ordered arrangement structure: (5-20) mL solution; and / or, During coaxial electrostatic spraying, the volume ratio of solution B to solution C is 1:(0.2~0.4); and / or, During uniaxial electrostatic spraying, the volume ratio of solution B to solution C is 1:(0.3~3).

14. The method for preparing the conjunctival scaffold according to claim 1, characterized in that: Step (4), During coaxial electrostatic spraying, the electrospraying rate of solution B is 0.1~10mL / h; the electrospraying rate of solution C is 0.1~5mL / h; the voltage is 8~30kV; the receiving distance is 8~30cm; the spraying time is 5~360min; In the case of single-axis electrostatic spraying, the propulsion rate of the electrospray solution after the mixture of solution B and solution C 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. The method for preparing the conjunctival scaffold according to claim 14, characterized in that: Step (4), During coaxial electrostatic spraying, the electrospraying rate of solution B is 1~3mL / h; the electrospraying rate of solution C is 0.4~1mL / h; the voltage is 15~25 kV; the receiving distance is 15~20cm; the spraying time is 15~120min; During uniaxial electrostatic spraying, the propulsion rate of the electrospray solution after the mixture of solution B and solution C is 0.5~3mL / h; the voltage is 15~25V; the receiving distance is 10~25cm; and the spraying time is 15~120min.

16. The method for preparing the conjunctival scaffold according to claim 15, characterized in that: Step (4), For coaxial electrostatic spraying, the spraying time is 15~40min; For single-axis electrostatic spraying, the spraying time is 15~60min.

17. A conjunctival stent prepared by the method according to any one of claims 1 to 16, characterized in that: The conjunctival support is a two-layer structure; The outer layer is electrospun fibers with a uniaxial orderly arrangement structure; The inner layer is an electrostatically sprayed layer loaded with active particles.

18. Use of a conjunctival scaffold prepared by the method according to any one of claims 1 to 16 in repairing conjunctival damage.

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