Artificial cornea and preparation method thereof

Abstract

The invention provides an artificial cornea and a preparation method thereof, and relates to the field of ophthalmology medical instruments. The preparation method of the artificial cornea comprises the following steps that an acidic electrolyte is provided, the acidic electrolyte contains collagen, chitosan and hydrogen peroxide, the collagen in the acidic electrolyte is pre-treated collagen, the pre-treatment comprises first treatment and second treatment, the first treatment comprises freeze-drying treatment on collagen liquid, the second treatment comprises freeze-drying treatment on the collagen liquid, and the third treatment comprises freeze-drying treatment on the collagen liquid; the collagen solution contains a phosphate solution, and collagen is dissolved in the phosphate solution; the second treatment comprises the step of carrying out reverse osmosis treatment on the collagen subjected to the first treatment by utilizing an acid solution; the negative electrode is used as a working electrode, and the working electrode is provided with a working surface with a spherical convex surface or a spherical concave surface; the working electrode and the positive electrode are inserted into the acidic electrolyte and powered on to perform electrochemical reaction, and a gel film is formed on the working surface of the working electrode. The artificial cornea prepared by the preparation method is relatively high in light transmittance and relatively good in mechanical strength.

Description

Technical Field

[0001] The present application relates to the field of ophthalmic medical devices, and more specifically, to an artificial cornea and a method for preparing the same. Background Art

[0002] Corneal diseases and cataracts are the main causes of blindness, and can be cured by corneal transplantation. However, the source of corneal donors is limited and cannot meet the demand. In order to alleviate the shortage of corneal donor sources, artificial corneas have emerged to replace donor corneas.

[0003] Existing artificial corneas are generally made of artificial synthetic materials, such as polyethylene glycol, polycaprolactone, polyhydroxyethyl methacrylate (PHEMA), polymethyl methacrylate (PMMA) and silicone gel. However, these synthetic materials often have poor biocompatibility with eye tissues and are easily rejected.

[0004] The main component of natural corneal stroma is collagen, which has better biocompatibility than synthetic materials. Based on this, the present application provides an artificial cornea using collagen as raw material and a preparation method thereof. Summary of the invention

[0005] The present application provides an artificial cornea and a preparation method thereof, and the prepared artificial cornea has a high light transmittance.

[0006] This application is implemented as follows:

[0007] In a first aspect, the present application provides a method for preparing an artificial cornea, comprising the following steps:

[0008] An acidic electrolyte is provided, wherein the acidic electrolyte contains collagen, chitosan and hydrogen peroxide, wherein the collagen in the acidic electrolyte is pre-treated collagen, wherein the pre-treatment includes a first treatment and a second treatment, wherein the first treatment includes freeze-drying the collagen solution, wherein the collagen solution contains a phosphate solution, and the collagen is dissolved in the phosphate solution; and the second treatment includes reverse osmosis treatment of the collagen after the first treatment using an acid solution;

[0009] The cathode electrode is used as the working electrode, wherein the working electrode has a spherical convex or concave working surface; the working electrode and the anode electrode are inserted into the acidic electrolyte and a power source is turned on to perform an electrochemical reaction, and a gel film is formed on the working surface of the working electrode.

[0010] In a possible embodiment, the step of performing reverse osmosis treatment on the first treated collagen using an acid solution comprises: mixing the first treated collagen with water and putting the mixture into a dialysis bag, and placing the dialysis bag in an acid solution dialysis external fluid for 1 to 5 days;

[0011] Optionally, the concentration of the acid solution is 0.0005-0.02 mol / L; optionally, the acid solution includes acetic acid solution.

[0012] In a possible implementation manner, the pH value of the acidic electrolyte is 3 to 6.5.

[0013] In a possible implementation manner, the mass ratio of the collagen to the chitosan in the acidic electrolyte is 65-115:80-100.

[0014] In a possible embodiment, the preparation step of the acidic electrolyte includes: mixing the collagen after the second treatment with a chitosan solution, adjusting the pH and mixing with a hydrogen peroxide solution to obtain the acidic electrolyte; optionally, the concentration of the collagen in the acidic electrolyte is 0.1 to 20 mg / mL, and the concentration of the chitosan is 0.5 to 10 mg / mL.

[0015] In one possible embodiment, the phosphate consists of monohydrogen phosphate and dihydrogen phosphate.

[0016] In a possible implementation manner, the pH value of the phosphate solution is 6-8.

[0017] The preparation step of the collagen solution includes: mixing the collagen solution after dialyzed and desalted with a phosphate solution so that the collagen is dissolved in the phosphate solution to obtain the collagen solution.

[0018] In a possible embodiment, the method further comprises cross-linking the gel film.

[0019] In a possible embodiment, the first treatment further comprises irradiating the freeze-dried collagen; optionally, the irradiation intensity of the irradiation treatment is 5 to 30 KGy.

[0020] In a second aspect, the present application provides an artificial cornea, which is prepared by the method for preparing an artificial cornea of ​​the first aspect.

[0021] This application has at least the following beneficial effects:

[0022] The preparation method of the artificial cornea of ​​the present application is that the collagen solution contains a phosphate solution and the collagen is dissolved in the phosphate solution to obtain a transparent collagen solution. The collagen solution is freeze-dried to break the collagen structure, and then the phosphate is replaced by reverse osmosis with an acid solution. The obtained acidic electrolyte is acidic. Collagen and chitosan are both positively charged in the acidic electrolyte. During the electrochemical reaction, the collagen and chitosan are deposited on the surface of the cathode to form a gel film. Due to the addition of chitosan, the collagen structure is interrupted during the pretreatment process after the collagen is treated with a phosphate solution and freeze-dried. During the electrochemical reaction, the collagen is more easily self-assembled to form uniformly arranged collagen fiber bundles, so the artificial cornea obtained has a higher light transmittance. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a photo of the artificial cornea prepared in Example 1;

[0024] Figure 2 This is a photo of the artificial cornea prepared in Example 2;

[0025] Figure 3 This is a photograph of the artificial cornea prepared in Example 9;

[0026] Figure 4 This is a photograph of the artificial cornea prepared in Example 12;

[0027] Figure 5 This is a photograph of the artificial cornea prepared in Comparative Example 4;

[0028] Figure 6 This is a photograph of the artificial cornea prepared in Comparative Example 9. DETAILED DESCRIPTION

[0029] The embodiments of the present application will be described in detail below in conjunction with the examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present application and should not be considered as limiting the scope of the present application. In the examples, if specific conditions are not specified, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0030] The following is a detailed description of the artificial cornea and its preparation method according to the embodiment of the present application:

[0031] In a first aspect, the present application provides an artificial cornea and a method for preparing the same, comprising the following steps:

[0032] An acidic electrolyte is provided, wherein the acidic electrolyte contains collagen, chitosan and hydrogen peroxide, wherein the collagen in the acidic electrolyte is pre-treated collagen, wherein the pre-treatment includes a first treatment and a second treatment; wherein the first treatment includes freeze-drying the collagen solution, wherein the collagen solution contains a phosphate solution, and the collagen is dissolved in the phosphate solution; wherein the second treatment includes treating the collagen after the first treatment with an acid solution; wherein the steps of preparing the collagen solution include: mixing the collagen solution after dialysis and desalination with a phosphate solution so that the collagen is dissolved in the phosphate solution, thereby obtaining the collagen solution;

[0033] The cathode electrode is used as the working electrode, wherein the working electrode has a spherical convex or spherical concave working surface; the working electrode and the anode electrode are inserted into an acidic electrolyte and powered on to perform an electrochemical reaction, and a gel film is formed on the working surface of the working electrode. Exemplarily, the anode electrode is a platinum electrode or a platinum-plated titanium electrode.

[0034] The preparation method of the artificial cornea of ​​the present application comprises the following steps: the collagen solution contains a phosphate solution and the collagen is dissolved in the phosphate solution to obtain a transparent collagen solution. The collagen solution is freeze-dried to break the collagen structure, and the phosphate is replaced by reverse osmosis treatment with an acid solution, so that the obtained acidic electrolyte is acidic. Collagen and chitosan are both positively charged in the acidic electrolyte. During the electrochemical reaction, the collagen and chitosan are deposited on the surface of the cathode electrode to form a gel film. Due to the addition of chitosan, the collagen structure is broken during the pretreatment process after the collagen is treated with a phosphate solution and freeze-dried. During the electrochemical reaction, the collagen is more likely to self-assemble to form uniformly arranged collagen fiber bundles, so that the obtained artificial cornea has a high light transmittance, with a light transmittance of more than 80% at 400nm and a light transmittance of more than 90% at 800nm. Moreover, after testing, the mechanical properties of the artificial cornea are also good.

[0035] Wherein, the preparation step of the collagen solution includes: mixing the collagen stock solution after dialysis and desalination with a phosphate solution so that the collagen is dissolved in the phosphate solution to obtain a collagen solution. The collagen stock solution is mixed with the phosphate solution, and the collagen is dissolved in the phosphate solution to form a transparent solution. The obtained collagen solution is then subjected to a first treatment and a second treatment, and the gel film finally formed has a good transmittance. Optionally, the phosphate is composed of monohydrogen phosphate and dihydrogen phosphate, for example, the phosphate buffer is composed of sodium monohydrogen phosphate and potassium dihydrogen phosphate; or it is composed of sodium monohydrogen phosphate and sodium dihydrogen phosphate; or it is composed of potassium monohydrogen phosphate and potassium dihydrogen phosphate. Optionally, the pH of the phosphate solution is 6 to 8, for example, the pH is any one of 6, 6.2, 6.4, 6.5, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8 and 8 or a value between any two.

[0036] Furthermore, the first treatment also includes irradiating the freeze-dried collagen. Irradiation can further disrupt the structure of the collagen molecules, making the collagen structure have a shorter molecular weight, and during electrochemical deposition, it is easier to self-assemble into a uniform and orderly structure, thereby forming a gel film with better light transmittance.

[0037] Optionally, the irradiation intensity of the irradiation treatment is 5 to 30 KGy, for example, any one of 5 KGy, 10 KGy, 15 KGy, 20 KGy, 25 KGy and 30 KGy, or a value between any two of them.

[0038] In addition, the step of using an acid solution to perform reverse osmosis treatment on the first treated collagen includes: mixing the first treated collagen with water and then putting it into a dialysis bag, and placing the dialysis bag in an acid solution dialysis external solution for treatment for 1 to 5 days. The first treated collagen contains phosphate. By mixing the first treated collagen with water and then putting it into a dialysis bag, and placing the dialysis bag in an acid solution for treatment, the acid solution can enter the dialysis bag and mix with the collagen, and the phosphate is replaced, so that the conductivity of the collagen solution is greatly reduced, which is conducive to obtaining an artificial cornea with better transparency. Moreover, the inventors of the present application found in their research that if the first treated collagen is directly mixed with an acid solution for treatment, the phosphate is not replaced, resulting in a high conductivity and a transparent artificial cornea cannot be formed.

[0039] Exemplarily, the acid solution includes acetic acid solution or propionic acid solution, and the concentration of the acid solution is 0.0005-0.02 mol / L. Optionally, the concentration of the acid solution is 0.0005 mol / L, 0.001 mol / L, 0.003 mol / L, 0.005 mol / L, 0.008 mol / L, 0.01 mol / L, 0.012 mol / L, 0.014 mol / L, 0.016 mol / L, 0.018 mol / L and 0.02 mol / L, or a value between any two thereof.

[0040] The preparation step of the acidic electrolyte includes: mixing the collagen after the second treatment with the chitosan solution, adjusting the pH and mixing with the hydrogen peroxide solution to obtain the acidic electrolyte. The pH value of the acidic electrolyte is 3 to 6.5, for example, the pH value of the acidic electrolyte is any one of 3, 3.5, 4, 4.5, 5, 5.5, 6 and 6.5 or any value between two of them.

[0041] Exemplarily, the concentration of collagen in the acidic electrolyte is 0.1 to 20 mg / mL, for example, any one of 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 3 mg / mL, 5 mg / mL, 7 mg / mL, 10 mg / mL, 12 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 18 mg / mL and 20 mg / mL, or a value between any two of them.

[0042] Exemplarily, the concentration of chitosan in the acidic electrolyte is 0.5 to 10 mg / mL, for example, any one of 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL and 10 mg / mL, or a value between any two of them.

[0043] Exemplarily, the concentration of hydrogen peroxide added is 3 to 50 μg / mL, for example, any one of 3 μg / mL, 5 μg / mL, 8 μg / mL, 10 μg / mL, 15 μg / mL, 20 μg / mL, 25 μg / mL, 30 μg / mL, 35 μg / mL, 40 μg / mL, 45 μg / mL and 50 μg / mL, or a value between any two of them.

[0044] Furthermore, the mass ratio of collagen to chitosan in the acidic electrolyte is 65-115:80-100. The inventors of the present application found in their research that the mass ratio of collagen to chitosan also affects the light transmittance of the artificial cornea, and when the mass ratio of collagen to chitosan is 65-115:80-100, the light transmittance of the artificial cornea can be further improved. Exemplarily, the mass ratio of collagen to chitosan in the acidic electrolyte is 66:80, 70:100, 80:100, 80:95, 87:95, 90:100, 95:100, 100:80, 105:90 or 112:100.

[0045] In addition, after the gel film is formed on the working surface of the working electrode, the gel film can also be cross-linked, so as to improve the mechanical properties of the artificial cornea. Optionally, the cross-linking treatment can be carried out by at least one of photochemical cross-linking, glutaraldehyde cross-linking and EDC-NHS cross-linking. Among them, glutaraldehyde cross-linking is to immerse the gel film in a glutaraldehyde solution, and then wash it alternately with physiological saline and pure water after the cross-linking treatment. When EDC-NHS cross-linking is adopted, the gel film is immersed in an EDC-NHS solution, and then wash it alternately with physiological saline and pure water after the cross-linking treatment. When photochemical cross-linking is adopted, the gel film is immersed in a photosensitizer, and then ultraviolet light is used for irradiation cross-linking. Optionally, riboflavin is selected as the photosensitizer.

[0046] In a second aspect, the present application provides an artificial cornea, which is prepared by the above-mentioned method for preparing an artificial cornea.

[0047] The artificial cornea prepared in the present application has a complete shape, good light transmittance, and high mechanical properties, and can replace the donor cornea for the treatment of corneal diseases.

[0048] The artificial cornea and the preparation method thereof of the present application are further described in detail below in conjunction with the embodiments.

[0049] Example 1

[0050] This embodiment provides a method for preparing an artificial cornea, which comprises the following steps:

[0051] (1) Providing a collagen stock solution after salting out and desalting, and mixing the collagen stock solution after salting out and desalting with a phosphate buffer solution, so that the collagen is dissolved in the phosphate solution to obtain a collagen solution. The phosphate buffer solution is composed of sodium monohydrogen phosphate and potassium dihydrogen phosphate, and has a pH of 6.8.

[0052] (2) freeze-drying the collagen obtained in step (1) to obtain freeze-dried collagen fibers, and then irradiating them to obtain irradiated freeze-dried collagen, wherein the irradiation intensity is 25 KGy.

[0053] (3) After mixing and stirring the irradiated freeze-dried collagen obtained in step (2) with water, the mixture is placed in a dialysis bag and placed in a dialysis external solution of 0.01 mol / L acetic acid solution for 3 days, with the solution being changed 3 times a day.

[0054] (4) taking the collagen treated in step (3), adding water and stirring evenly, then adding chitosan solution, mixing to obtain 40 mL of a mixed solution, stirring evenly, adjusting the pH to 4.5, and then adding hydrogen peroxide at a ratio of 10 μL / mL to obtain an acidic electrolyte. The content of collagen in the acidic electrolyte is 87 mg, and the content of chitosan is 100 mg.

[0055] (5) The cathode electrode is used as the working electrode. The working electrode is made of titanium and has a spherical convex working surface. The platinum sheet is used as the anode electrode. The anode electrode has a spherical convexity and is arranged in alignment with the spherical convexity of the cathode electrode. The curvature of the cathode electrode and the anode electrode is 8. The working electrode and the anode electrode are placed horizontally and inserted into the acidic electrolyte, and the distance between them is controlled to be 8 cm. Then, a power supply is turned on and a voltage of 5 V is applied to allow the electrochemical reaction to proceed for 20 minutes, and a gel film is formed on the working surface of the working electrode. Among them, the artificial cornea prepared in this embodiment is as follows Figure 1 shown.

[0056] Example 2

[0057] This embodiment provides a method for preparing an artificial cornea. Compared with embodiment 1, the only difference is that this embodiment omits the irradiation treatment step in step (2) of embodiment 1, and step (3) adopts freeze-dried collagen and water mixed and stirred, and then put into a dialysis bag. Among them, the artificial cornea prepared in this embodiment is as follows Figure 2 shown.

[0058] Example 3

[0059] This embodiment provides a method for preparing an artificial cornea. Compared with Embodiment 1, the only difference is that in this embodiment, the content of collagen in the electrolyte is 80 mg, and the content of chitosan is 100 mg.

[0060] Example 4

[0061] This embodiment provides a method for preparing an artificial cornea. Compared with Embodiment 1, the only difference is that in this embodiment, the content of collagen in the electrolyte is 103 mg.

[0062] Example 5

[0063] This embodiment provides a method for preparing an artificial cornea. Compared with Embodiment 1, the only difference is that in this embodiment, the content of collagen in the electrolyte is 80 mg, and the content of chitosan is 95 mg.

[0064] Example 6

[0065] This embodiment provides a method for preparing an artificial cornea. Compared with Embodiment 1, the only difference is that in this embodiment, the content of collagen in the electrolyte is 66 mg, and the content of chitosan is 80 mg.

[0066] Example 7

[0067] This embodiment provides a method for preparing an artificial cornea. Compared with Embodiment 1, the only difference is that in this embodiment, the content of collagen in the electrolyte is 112 mg, and the content of chitosan is 100 mg.

[0068] Example 8

[0069] This embodiment provides a method for preparing an artificial cornea. Compared with Embodiment 1, the only difference is that in this embodiment, the content of collagen in the electrolyte is 32 mg, and the content of chitosan is 100 mg.

[0070] Example 9

[0071] This embodiment provides a method for preparing an artificial cornea. Compared with the embodiment 1, the only difference is that in this embodiment, the voltage during electrochemical deposition is 9V. The results show that the artificial cornea prepared in the embodiment 9 is easy to produce radial lines (such as Figure 3 shown).

[0072] Example 10

[0073] This embodiment provides a method for preparing an artificial cornea. Compared with Embodiment 1, the only difference is that in this embodiment, the current during electrochemical deposition is 15 mA.

[0074] Embodiment 11

[0075] This embodiment provides a method for preparing an artificial cornea. Compared with Embodiment 1, the only difference is that in this embodiment, the content of collagen in the electrolyte is 52.5 mg, and the content of chitosan is 60 mg.

[0076] Example 12

[0077] This embodiment provides a method for preparing an artificial cornea. Compared with Embodiment 1, the only difference is that in this embodiment, the content of collagen in the electrolyte is 32 mg, and the content of chitosan is 40 mg. The artificial cornea prepared in this embodiment is as follows: Figure 4 shown.

[0078] Embodiment 13

[0079] This embodiment provides a method for preparing an artificial cornea, which is different from the embodiment 1 only in that it further includes cross-linking the gel film of the embodiment 1, and the cross-linking step includes: soaking the gel film in an EDC-NHS solution for 20 minutes, wherein the concentration of the EDC cross-linking agent is 20 mg / mL, and the molar ratio of the EDC cross-linking agent to the NHS cross-linking agent is 2: 1. After the cross-linking treatment, the gel film is alternately washed with physiological saline and pure water.

[0080] Embodiment 14

[0081] This embodiment provides a method for preparing an artificial cornea. Compared with Embodiment 1, the only difference is that the pH value of the phosphate buffer solution is 6.

[0082] Embodiment 15

[0083] This embodiment provides a method for preparing an artificial cornea. Compared with Embodiment 1, the only difference is that the pH of the phosphate buffer is 7.8.

[0084] Comparative Example 1

[0085] Comparative Example 1 provides a method for preparing an artificial cornea. Compared with Example 1, the only difference is that Comparative Example 1 omits step (3) in Example 1, and the collagen used in step (4) is the irradiated freeze-dried collagen treated in step (2). The results show that after the electrochemical reaction, no gel film is formed on the surface of the cathode electrode.

[0086] Comparative Example 2

[0087] Comparative Example 2 provides a method for preparing an artificial cornea. Compared with Example 1, the only difference is that Comparative Example 2 omits the irradiation treatment step and step (3) in step (2) of Example 1, and the collagen used in step (4) is the freeze-dried collagen formed in step (2). During the electrodeposition, a small amount of bubbles are generated, and although there is deposition on the surface of the cathode electrode, it is not formed.

[0088] Comparative Example 3

[0089] Comparative Example 3 provides a method for preparing an artificial cornea, which comprises the following steps:

[0090] A collagen stock solution after salting out and desalting is provided, and the collagen stock solution is mixed with a phosphate buffer solution, so that the collagen is dissolved in the phosphate solution to obtain a collagen solution, wherein the phosphate buffer solution is composed of sodium monohydrogen phosphate and potassium dihydrogen phosphate, and has a pH of 6.8.

[0091] 40 mL of collagen solution (collagen content: 87 mg) was mixed with 400 μL of chitosan solution (chitosan content: 100 mg). During the mixing, flocculent precipitation was generated, which could not be redispersed by stirring, and the subsequent electrodeposition reaction could not be carried out.

[0092] Comparative Example 4

[0093] Comparative Example 4 provides a method for preparing an artificial cornea. Compared with Example 1, the only difference is that Comparative Example 4 omits step (1) of Example 1, and step (2) uses the lyophilized and desalted collagen stock solution to perform irradiation.

[0094] The artificial cornea prepared in Comparative Example 4 is as follows Figure 5 As shown, the overall appearance is milky white with poor transparency.

[0095] Comparative Example 5

[0096] Comparative Example 5 provides a method for preparing an artificial cornea. Compared with Example 1, the only difference is that no chitosan solution is added in step (4) of Comparative Example 5, and the content of collagen is 187 mg. The results show that the artificial cornea prepared in Comparative Example 5 is relatively soft and has poor tensile strength.

[0097] Comparative Example 6

[0098] Comparative Example 6 provides a method for preparing an artificial cornea. Compared with Comparative Example 5, the only difference is that in Comparative Example 6, the collagen stock solution after dialysis and desalination is freeze-dried.

[0099] The results showed that the artificial cornea prepared in Comparative Example 6 could be formed and had a softer texture than the artificial cornea in Example 1, and was damaged during the process of being removed from the electrode.

[0100] Comparative Example 7

[0101] Comparative Example 7 provides a method for preparing an artificial cornea. Compared with Example 6, the only difference is that Comparative Example 7 omits step (1) and step (3) of Example 6, step (2) uses the collagen stock solution after dialysis and desalination to be freeze-dried and irradiated, and the collagen used in step (4) is the freeze-dried irradiated collagen treated in step (2).

[0102] Comparative Example 8

[0103] Comparative Example 8 provides a method for preparing an artificial cornea, which comprises the following steps:

[0104] (1) A dialyzed desalted collagen stock solution is provided, 40 mL of the dialyzed desalted collagen stock solution is adjusted to pH 4.5, and then hydrogen peroxide is added at a ratio of 10 μL / mL to obtain an acidic electrolyte, wherein the collagen content in the acidic electrolyte is 187 mg.

[0105] (2) The cathode electrode is used as the working electrode. The working electrode is made of titanium and has a spherical convex working surface. The platinum sheet is used as the anode electrode. The anode electrode has a spherical convexity and is arranged in alignment with the cathode electrode. The curvature of the cathode electrode and the anode electrode is 8. The working electrode and the anode electrode are placed horizontally and inserted into the acidic electrolyte, and the distance between them is controlled to be 8 cm. Then, a power supply is turned on and a voltage of 5 V is applied to allow the electrochemical reaction to proceed for 20 minutes. The results show that a large number of bubbles are generated during the electrochemical process, and no gel film is deposited on the surface of the cathode electrode.

[0106] Comparative Example 9

[0107] Comparative Example 9 provides a method for preparing an artificial cornea. Compared with Example 1, the only difference is that Comparative Example 9 omits the freeze-drying and irradiation steps in step (2) of Example 1, and the collagen used in step (3) is the collagen solution treated in step (1).

[0108] The results show that although the artificial cornea prepared in Comparative Example 9 is formed, some white spots can be clearly seen, and the whole is not uniform and transparent (such as Figure 6 shown).

[0109] Test Example 1

[0110] The light transmittance of the artificial corneas prepared in Examples 1 to 10, Examples 13 to 15, and Comparative Examples 4 and 7 was tested. The test results of the artificial corneas at 400 nm and 800 nm are shown in Table 1.

[0111] Table 1. Transmittance test results

[0112]

[0113]

[0114] From the results in Table 1, it can be seen that the artificial corneas prepared in Examples 1 to 10 and Examples 13 to 15 of the present application have a transmittance at 400nm that is greater than 80%, and a transmittance at 800nm ​​that is greater than 90%, indicating that the artificial corneas prepared in the examples of the present application have a high transmittance, which is equivalent to the transmittance of the human cornea.

[0115] By comparing Example 1 with Comparative Example 4, in the preparation method of Comparative Example 4, the collagen was not treated with a phosphate solution, and the transmittance of the artificial cornea at 400nm was less than 70%, indicating that the use of phosphate treatment can improve the transmittance of the artificial cornea at 400nm. In addition, by comparing Example 6 with Comparative Example 7, in the preparation method of Comparative Example 7, the collagen was not treated with a phosphate buffer solution and was not treated with reverse osmosis, and the transmittance of the artificial cornea at 400nm was less than 60%. It shows that the collagen is treated with a phosphate solution and then treated with reverse osmosis, which is beneficial to improve the transmittance at 400nm.

[0116] By comparing Example 1 and Example 10, it is found that the transmittance of the artificial cornea prepared in Example 10 at 400nm is greater than 80%, and the transmittance at 800nm ​​is greater than 90%, but the transmittance of the artificial cornea prepared in Example 10 at 400nm and 800nm ​​is also worse than that of the artificial cornea prepared in Example 1, indicating that it is easier to obtain an artificial cornea with high transmittance by using a constant voltage method than a constant current method. In addition, the artificial cornea prepared in Example 9 has radial lines, indicating that excessive voltage can easily lead to slight lines on the artificial cornea.

[0117] By comparing Example 1, Example 2 and Comparative Examples 1 to 3, Comparative Example 1 did not form a gel film on the surface of the cathode electrode; although Comparative Example 2 had deposition on the surface of the cathode electrode, it was not formed; in Comparative Example 3, when the collagen solution and the chitosan solution were mixed, flocculent sedimentation occurred, and the subsequent electrochemical reaction could not be carried out. It is explained that simply phosphate treatment of collagen, or phosphate treatment and freeze-drying of collagen, or phosphate treatment, freeze-drying and irradiation of collagen will result in the inability to form a gel film. However, by adopting the preparation process of Examples 1 and 2 of the present application, phosphate treatment, freeze-drying and reverse osmosis treatment of collagen, or further irradiation treatment after freeze-drying the collagen, an artificial cornea with good light transmittance can be formed.

[0118] In addition, by comparing Example 1, Example 2 and Comparative Example 8, Comparative Example 8 uses the collagen stock solution after dialysis and desalination to mix with hydrogen peroxide, the collagen has not been pre-treated as specified in the present application, and no chitosan solution is added to the electrolyte. The results show that a large number of bubbles are generated during the electrochemical reaction, and no gel film is deposited on the surface of the cathode electrode. This shows that the pre-treatment of collagen and the addition of chitosan as specified in the present application can be conducive to the preparation of an artificial cornea with good light transmittance.

[0119] By comparing Example 1 and Example 13, Example 13 is a cross-linked artificial cornea, while Example 1 is not cross-linked, and the light transmittance of the artificial cornea of ​​Example 13 is not much different from that of Example 1. This indicates that cross-linking the artificial cornea with the EDC-NHS cross-linking agent will not substantially affect the light transmittance of the artificial cornea.

[0120] Among them, the inventors of the present application found in the experiment that the artificial corneas prepared in Example 8, Example 11 and Example 12 were transparent, but showed a slightly collapsed state after being removed from the surface of the cathode electrode, and were more likely to cause cracks during the removal process. This shows that the concentration of collagen and chitosan in the electrolyte will affect the mechanical properties of the artificial cornea. When the concentration of collagen and / or chitosan in the electrolyte is low, the artificial cornea will become soft and brittle.

[0121] Test Example 2

[0122] The artificial corneas prepared in Example 1, Example 2, and Comparative Example 5 were cross-linked for 20 minutes using an EDC-NHS cross-linking agent, wherein the concentration of the EDC cross-linking agent was 20 mg / mL, and the molar ratio of the EDC cross-linking agent to the NHS cross-linking agent was 2:1. The samples were made into long strips for mechanical property testing, and the results are shown in Table 2.

[0123] Table 2. Mechanical properties test results

[0124] Tensile strength / MPa Suture strength / N Example 1 0.58 0.06 Example 2 0.52 —— Comparative Example 5 0.14 ——

[0125] From the results in Table 2, it can be seen that the tensile strength of the artificial corneas prepared in Examples 1 and 2 of the present application is greater than that of the artificial cornea of ​​Comparative Example 5, indicating that the addition of chitosan can improve the tensile strength of the artificial cornea. In addition, the artificial cornea prepared in Example 1 of the present application has good suture strength.

[0126] The above description is only a specific embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing an artificial cornea, characterized in that: The following steps are involved: An acidic electrolyte is provided, wherein the acidic electrolyte contains collagen, chitosan and hydrogen peroxide, wherein the collagen in the acidic electrolyte is pre-treated collagen, wherein the pre-treatment includes a first treatment and a second treatment, wherein the first treatment includes freeze-drying the collagen solution, wherein the collagen solution contains a phosphate solution, and the collagen is dissolved in the phosphate solution; and the second treatment includes reverse osmosis treatment of the collagen after the first treatment using an acid solution; The cathode electrode is used as the working electrode, wherein the working electrode has a spherical convex or concave working surface; the working electrode and the anode electrode are inserted into the acidic electrolyte and a power source is turned on to perform an electrochemical reaction, and a gel film is formed on the working surface of the working electrode.

2. The method for preparing an artificial cornea according to claim 1, characterized in that: The step of performing reverse osmosis treatment on the first treated collagen using an acid solution comprises: mixing the first treated collagen with water and putting the mixture into a dialysis bag, and placing the dialysis bag in an acid solution dialysis external fluid for 1 to 5 days; Optionally, the concentration of the acid solution is 0.0005-0.02 mol / L; optionally, the acid solution includes acetic acid solution.

3. The method for preparing an artificial cornea according to claim 1, characterized in that: The pH value of the acidic electrolyte is 3 to 6.

5.

4. The method for preparing an artificial cornea according to claim 1, characterized in that: The mass ratio of the collagen to the chitosan in the acidic electrolyte is 65-115:80-100.

5. The method for preparing an artificial cornea according to claim 1, characterized in that: The preparation step of the acidic electrolyte comprises: mixing the collagen after the second treatment with a chitosan solution, adjusting the pH and mixing with a hydrogen peroxide solution to obtain the acidic electrolyte; optionally, the concentration of the collagen in the acidic electrolyte is 0.1 to 20 mg / mL, and the concentration of the chitosan is 0.5 to 10 mg / mL.

6. The method for preparing an artificial cornea according to any one of claims 1 to 5, characterized in that: The phosphate consists of monohydrogen phosphate and dihydrogen phosphate; optionally, the pH value of the phosphate solution is 6-8.

7. The method for preparing an artificial cornea according to claim 6, characterized in that: The preparation step of the collagen solution includes: mixing the collagen solution after dialyzed and desalted with a phosphate solution so that the collagen is dissolved in the phosphate solution to obtain the collagen solution.

8. The method for preparing an artificial cornea according to any one of claims 1 to 5, characterized in that: The method also includes cross-linking the gel film.

9. The method for preparing an artificial cornea according to any one of claims 1 to 5, characterized in that: The first treatment further comprises irradiating the freeze-dried collagen; optionally, the irradiation intensity of the irradiation treatment is 5 to 30 KGy.

10. An artificial cornea, characterized in that: The artificial cornea is prepared by the method for preparing the artificial cornea according to any one of claims 1 to 9.

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