An artificial cornea and a method of making the same

Abstract

This application provides an artificial cornea and its preparation method, relating to the field of ophthalmic medical devices. The method for preparing the artificial cornea includes the following steps: providing an acidic electrolyte containing collagen, chitosan, and hydrogen peroxide; the collagen in the acidic electrolyte is pretreated collagen, the pretreatment including a first treatment and a second treatment; the first treatment includes freeze-drying the collagen solution, the collagen solution containing a phosphate solution, and the collagen dissolved in the phosphate solution; the second treatment includes reverse osmosis treatment of the first-treated collagen using an acidic solution; using a negative electrode as the working electrode, wherein the working electrode has a spherically convex or spherically concave working surface; inserting the working electrode and the positive electrode into the acidic electrolyte and applying a power source to induce an electrochemical reaction, forming a gel film on the working surface of the working electrode. The artificial cornea prepared by this method has high light transmittance and good mechanical strength.

Description

Technical Field

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

[0002] Corneal diseases and cataracts are leading causes of blindness, which can be cured through corneal transplantation. However, the supply of corneal donors is limited and cannot meet the demand. To alleviate the shortage of corneal donors, artificial corneas have been developed to replace donor corneas.

[0003] Existing artificial corneas are generally made of 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 prone to rejection.

[0004] The main component of the natural corneal stroma is collagen, which has better biocompatibility compared to synthetic materials. Based on this, this application provides an artificial cornea made from collagen and a method for its preparation. Summary of the Invention

[0005] This application provides an artificial cornea and a method for preparing the same, resulting in an artificial cornea with high light transmittance.

[0006] This application is implemented as follows:

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

[0008] An acidic electrolyte is provided, the acidic electrolyte containing collagen, chitosan, and hydrogen peroxide, wherein the collagen in the acidic electrolyte is pretreated collagen, the pretreatment including a first treatment and a second treatment; the first treatment includes lyophilizing the collagen solution, the collagen solution containing a phosphate solution, and the collagen dissolved in the phosphate solution; the second treatment includes reverse osmosis treatment of the collagen after the first treatment using an acid solution.

[0009] The cathode 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 are inserted into the acidic electrolyte and a power source is applied to carry out an electrochemical reaction, and a gel film is formed on the working surface of the working electrode.

[0010] In one possible implementation, the step of reverse osmosis treatment of the first-treated collagen with an acid solution includes: mixing the first-treated collagen with water and placing it into a dialysis bag, and then placing the dialysis bag in an acid solution dialysis fluid for 1 to 5 days.

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

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

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

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

[0015] In one possible implementation, the phosphate is composed of monohydrogen phosphate and dihydrogen phosphate.

[0016] In one possible implementation, the pH of the phosphate solution is 6 to 8.

[0017] The preparation steps of the collagen solution include: mixing the collagen stock solution after dialysis and desalting with a phosphate solution to dissolve the collagen in the phosphate solution, thereby obtaining the collagen solution.

[0018] In one possible implementation, the gel membrane is further subjected to a crosslinking treatment.

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

[0020] Secondly, this application provides an artificial cornea, which is prepared by the method for preparing an artificial cornea according to the first aspect.

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

[0022] The method for preparing an artificial cornea disclosed in this application involves a collagen solution containing a phosphate solution, which dissolves the collagen in the phosphate solution to obtain a transparent collagen solution. By freeze-drying the collagen solution, the collagen structure can be broken down. Then, the phosphate is replaced by reverse osmosis treatment with an acid solution, resulting in an acidic electrolyte. Both collagen and chitosan are positively charged in the acidic electrolyte. During the electrochemical reaction, 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 broken down during the pretreatment process after treatment with phosphate solution and freeze-drying. During the electrochemical reaction, the collagen is more likely to self-assemble into uniformly arranged collagen fiber bundles, thus resulting in artificial corneas with high light transmittance. Attached Figure Description

[0023] Figure 1 A photograph of the artificial cornea prepared in Example 1;

[0024] Figure 2 A photograph of the artificial cornea obtained in Example 2;

[0025] Figure 3 A photograph of the artificial cornea obtained in Example 9;

[0026] Figure 4 A photograph of the artificial cornea obtained in Example 12;

[0027] Figure 5 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 Implementation

[0029] The embodiments of this application will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of this application. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0030] The artificial cornea and its preparation method according to embodiments of this application are described in detail below:

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

[0032] An acidic electrolyte is provided, containing collagen, chitosan, and hydrogen peroxide. The collagen in the acidic electrolyte is pretreated collagen, which includes a first treatment and a second treatment. The first treatment includes lyophilizing the collagen solution, which contains a phosphate solution, and the collagen is dissolved in the phosphate solution. The second treatment includes treating the collagen after the first treatment with an acid solution. The preparation steps of the collagen solution include mixing the dialyzed and desalted collagen stock solution with a phosphate solution to dissolve the collagen in the phosphate solution, thereby obtaining the collagen solution.

[0033] The cathode is used as the working electrode, which has a spherical convex or spherical concave working surface. The working electrode and the anode are inserted into an acidic electrolyte and a power source is applied to induce an electrochemical reaction, forming a gel film on the working surface of the working electrode. For example, the anode is a platinum electrode or a platinum-plated titanium electrode.

[0034] The method for preparing an artificial cornea disclosed in this application involves a collagen solution containing a phosphate solution, which dissolves the collagen in the phosphate solution to obtain a transparent collagen solution. The collagen solution is then freeze-dried to break down the collagen structure. Next, reverse osmosis treatment with an acid solution is used to displace the phosphate, resulting in an acidic electrolyte. Both collagen and chitosan are positively charged in the acidic electrolyte. During the electrochemical reaction, collagen and chitosan deposit on the surface of the cathode to form a gel film. Due to the addition of chitosan, the collagen structure is broken down during the pretreatment process after phosphate solution treatment and freeze-drying. During the electrochemical reaction, the collagen more easily self-assembles into uniformly arranged collagen fiber bundles, resulting in artificial corneas with high light transmittance, exceeding 80% at 400 nm and exceeding 90% at 800 nm. Furthermore, tests have shown that the artificial cornea also exhibits good mechanical properties.

[0035] The preparation steps of the collagen solution include: mixing the dialyzed and desalted collagen stock solution with a phosphate solution to dissolve the collagen in the phosphate solution, thereby obtaining the collagen solution. The mixing of the collagen stock solution and the phosphate solution, with the collagen dissolving in the phosphate solution, forms a transparent solution. The obtained collagen solution undergoes a first treatment and a second treatment, resulting in a gel membrane with good light transmittance. Optionally, the phosphate is composed of monohydrogen phosphate and dihydrogen phosphate, for example, a phosphate buffer solution composed of sodium monohydrogen phosphate and potassium dihydrogen phosphate; or composed of sodium monohydrogen phosphate and sodium dihydrogen phosphate; or composed of potassium monohydrogen phosphate and potassium dihydrogen phosphate. Optionally, the pH of the phosphate solution is 6–8, for example, a value between or between any two of the following: pH 6, 6.2, 6.4, 6.5, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, and 8.

[0036] Furthermore, the first treatment also includes irradiating the freeze-dried collagen. Irradiation can further break down the structure of collagen molecules, resulting in a shorter molecular weight collagen structure. During electrochemical deposition, it is easier for the collagen to self-assemble into a uniform and ordered 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 or any two of 5 kGy, 10 kGy, 15 kGy, 20 kGy, 25 kGy and 30 kGy.

[0038] Furthermore, the step of reverse osmosis treatment of the first-treated collagen with an acid solution includes: mixing the first-treated collagen with water and placing it into a dialysis bag, then immersing the dialysis bag in the acid solution for 1-5 days. The first-treated collagen contains phosphates. By mixing the first-treated collagen with water and then placing it into a dialysis bag, followed by immersion in an acid solution, the acid solution can enter the dialysis bag and mix with the collagen, displacing the phosphates and significantly reducing the conductivity of the collagen solution, thus facilitating the formation of a transparent artificial cornea. Moreover, the inventors of this application discovered in their research that if the first-treated collagen is directly mixed with the acid solution, the phosphates are not displaced, resulting in high conductivity and preventing the formation of a transparent artificial cornea.

[0039] For example, the acid solution includes an acetic acid solution or a propionic acid solution, and the concentration of the acid solution is 0.0005 to 0.02 mol / L. Optionally, the concentration of the acid solution is any one or a value between any two of 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.

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

[0041] For example, the concentration of collagen in the acidic electrolyte is 0.1 to 20 mg / mL, such as any one or any two 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.

[0042] For example, the concentration of chitosan in the acidic electrolyte is 0.5 to 10 mg / mL, such as any one or any two 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.

[0043] For example, the concentration of added hydrogen peroxide is 3 to 50 μg / mL, such as any one or any combination 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.

[0044] Furthermore, the mass ratio of collagen to chitosan in the acidic electrolyte is 65–115:80–100. The inventors of this application discovered in their research that the mass ratio of collagen to chitosan also affects the light transmittance of the artificial cornea, and that when the mass ratio is 65–115:80–100, the light transmittance of the artificial cornea can be further improved. For example, 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 a gel film is formed on the working surface of the working electrode, the gel film can be cross-linked to improve the mechanical properties of the artificial cornea. Optionally, the cross-linking treatment can be performed using at least one of photochemical cross-linking, glutaraldehyde cross-linking, and EDC-NHS cross-linking. Specifically, glutaraldehyde cross-linking involves immersing the gel film in a glutaraldehyde solution, followed by alternating washing with physiological saline and pure water. EDC-NHS cross-linking involves immersing the gel film in an EDC-NHS solution, followed by alternating washing with physiological saline and pure water. Photochemical cross-linking involves immersing the gel film in a photosensitizer, followed by cross-linking using ultraviolet light; optionally, riboflavin is selected as the photosensitizer.

[0046] Secondly, this application provides an artificial cornea, which is prepared by the above-described method for preparing an artificial cornea.

[0047] The artificial cornea produced in this 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 its preparation method of this application are further described in detail below with reference to the embodiments.

[0049] Example 1

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

[0051] (1) Provide the collagen stock solution after salting out and desalting. Mix the collagen stock solution after salting out and desalting with phosphate buffer solution to dissolve the collagen in the phosphate solution to obtain the collagen solution. The phosphate buffer solution is composed of sodium monohydrogen phosphate and potassium dihydrogen phosphate, and the pH is 6.8.

[0052] (2) The collagen obtained in step (1) is freeze-dried to obtain freeze-dried collagen fibers, and then subjected to irradiation treatment to obtain irradiated freeze-dried collagen. The irradiation intensity is 25 KGy.

[0053] (3) After mixing and stirring the irradiated freeze-dried collagen obtained in step (2) with water, put it into a dialysis bag and place it in a 0.01 mol / L acetic acid solution for dialysis for 3 days, changing the solution 3 times a day.

[0054] (4) Take the collagen treated in step (3), add water and stir evenly, then add chitosan solution, mix to obtain 40 mL of mixture, stir evenly and adjust pH to 4.5, then add hydrogen peroxide at a ratio of 10 μL / mL to obtain acidic electrolyte. The collagen content in the acidic electrolyte is 87 mg and the chitosan content is 100 mg.

[0055] (5) Using a cathode as the working electrode, the working electrode is made of titanium and has a spherically convex working surface. A platinum sheet is used as the anode, which also has a spherically convex surface aligned with the spherically convex surface of the cathode. The curvature of both the cathode and anode is 8°. The working electrode and anode are horizontally inserted into an acidic electrolyte, with the distance between them controlled at 8 cm. A 5V voltage is then applied to allow an electrochemical reaction to occur for 20 minutes, forming a gel film on the working surface of the working electrode. The artificial cornea prepared in this embodiment is as follows: Figure 1 As shown.

[0056] Example 2

[0057] This embodiment provides a method for preparing an artificial cornea. Compared with Embodiment 1, the only difference is that the irradiation treatment step in step (2) of Embodiment 1 is omitted in this embodiment. Step (3) involves mixing and stirring freeze-dried collagen with water, and then placing the mixture into a dialysis bag. The artificial cornea prepared in this embodiment is as follows: Figure 2 As shown.

[0058] Example 3

[0059] This embodiment provides a method for preparing an artificial cornea. The only difference from Embodiment 1 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. The only difference from Embodiment 1 is that the collagen content in the electrolyte is 103 mg in this embodiment.

[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. The only difference from Embodiment 1 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. The only difference from Example 1 is that the voltage during electrochemical deposition is 9V. Results show that the artificial cornea prepared in Example 9 easily produces radial patterns (such as...). Figure 3 (As shown).

[0072] Example 10

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

[0074] Example 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 to 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 As shown.

[0078] Example 13

[0079] This embodiment provides a method for preparing an artificial cornea. Compared with Embodiment 1, the only difference is that it further includes crosslinking the gel membrane of Embodiment 1. The crosslinking step includes: immersing the gel membrane in an EDC-NHS solution for 20 minutes, wherein the concentration of the EDC crosslinking agent is 20 mg / mL, and the molar ratio of the EDC crosslinking agent to the NHS crosslinking agent is 2:1. After the crosslinking treatment, the membrane is alternately washed with physiological saline and pure water.

[0080] Example 14

[0081] This embodiment provides a method for preparing an artificial cornea, which differs from Embodiment 1 only in that the pH of the phosphate buffer solution is 6.

[0082] Example 15

[0083] This embodiment provides a method for preparing an artificial cornea. The only difference from Embodiment 1 is that the pH of the phosphate buffer solution 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) of Example 1, and the collagen used in step (4) is irradiated freeze-dried collagen treated in step (2). The results show that no gel film is formed on the surface of the cathode after the electrochemical reaction.

[0086] Comparative Example 2

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

[0088] Comparative Example 3

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

[0090] The collagen stock solution, after salting out and desalting, is mixed with a phosphate buffer solution to dissolve the collagen in the phosphate solution, thus obtaining the collagen solution. The phosphate buffer solution consists of sodium monohydrogen phosphate and potassium dihydrogen phosphate, with a pH of 6.8.

[0091] 40 mL of collagen solution (containing 87 mg of collagen) was mixed with 400 μL of chitosan solution (containing 100 mg of chitosan). During mixing, flocculent precipitate was formed and could not be redispersed by stirring, thus preventing subsequent electrodeposition reaction.

[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 collagen stock solution after dialysis and desalting to freeze-dry and irradiate.

[0094] The artificial cornea prepared in Comparative Example 4 is as follows: Figure 5 As shown, it is milky white overall and has 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 chitosan solution was not added in step (4) of Comparative Example 5, and the collagen content was 187 mg. The results showed that the artificial cornea prepared in Comparative Example 5 was softer and had poorer tensile strength.

[0097] Comparative Example 6

[0098] Comparative Example 6 provides a method for preparing an artificial cornea. The only difference between Comparative Example 6 and Comparative Example 5 is that Comparative Example 6 uses collagen stock solution after dialysis and desalting for freeze drying.

[0099] The results showed that the artificial cornea prepared in Comparative Example 6 could be shaped, had a softer texture than the artificial cornea in Example 1, and broke during removal from the electrodes.

[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 steps (1) and (3) of Example 6. In step (2), collagen stock solution after dialysis and desalting is freeze-dried and irradiated. In step (4), the collagen used is freeze-dried and irradiated collagen after step (2).

[0102] Comparative Example 8

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

[0104] (1) Provide the collagen stock solution after dialysis and desalting. Adjust the pH of 40 mL of the collagen stock solution after dialysis and desalting to 4.5, and then add hydrogen peroxide at a ratio of 10 μL / mL to obtain an acidic electrolyte. The collagen content in the acidic electrolyte is 187 mg.

[0105] (2) The cathode was used as the working electrode, which was made of titanium and had a spherically convex working surface. A platinum sheet was used as the anode, which also had a spherically convex surface and was aligned with the cathode. The curvature of both the cathode and anode was 8°. The working electrode and anode were horizontally inserted into the acidic electrolyte, with the distance between them controlled at 8 cm. A power supply of 5V was then applied to allow the electrochemical reaction to proceed for 20 min. The results showed that a large number of bubbles were generated during the electrochemical process, and no gel film was deposited on the surface of the cathode.

[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 showed that although the artificial cornea prepared in Comparative Example 9 was formed, some white spots were clearly visible, and the overall appearance was not uniformly transparent (e.g., Figure 6 (As shown).

[0109] Experimental Example 1

[0110] The 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 400nm and 800nm ​​are shown in Table 1.

[0111] Table 1. Transmittance Test Results

[0112]

[0113]

[0114] As can be seen from the results in Table 1, the artificial corneas prepared in Examples 1-10 and Examples 13-15 of this application all have a transmittance of more than 80% at 400nm and a transmittance of more than 90% at 800nm, indicating that the artificial corneas prepared in the examples of this application have high transmittance, which is comparable to that of human corneas.

[0115] Comparing Example 1 and Comparative Example 4, in the preparation method of Comparative Example 4, the collagen was not treated with phosphate solution, and the resulting artificial cornea had a transmittance of less than 70% at 400 nm, indicating that phosphate treatment can improve the transmittance of the artificial cornea at 400 nm. Furthermore, comparing Example 6 and Comparative Example 7, in the preparation method of Comparative Example 7, the collagen was not treated with phosphate buffer solution nor subjected to reverse osmosis, and the resulting artificial cornea had a transmittance of less than 60% at 400 nm. This indicates that phosphate solution treatment followed by reverse osmosis treatment of the collagen is beneficial for improving the transmittance at 400 nm.

[0116] A comparison of Examples 1 and 10 revealed that the artificial cornea prepared in Example 10 had a transmittance greater than 80% at 400 nm and greater than 90% at 800 nm. However, the transmittance of the artificial cornea prepared in Example 10 at both 400 nm and 800 nm was also lower than that of the artificial cornea prepared in Example 1, indicating that a constant voltage method is more likely to produce an artificial cornea with high transmittance than a constant current method. Furthermore, the artificial cornea prepared in Example 9 exhibited radial patterns, indicating that excessive voltage can easily lead to slight wrinkles in the artificial cornea.

[0117] By comparing Examples 1, 2, and Comparative Examples 1 to 3, it was found that Comparative Example 1 did not form a gel film on the surface of the cathode; Comparative Example 2 had deposition on the surface of the cathode, but it did not form a gel film; and in Comparative Example 3, flocculent sedimentation occurred when the collagen solution and chitosan solution were mixed, preventing subsequent electrochemical reactions. This indicates that simple phosphate treatment of collagen, phosphate treatment and freeze-drying of collagen, or phosphate treatment, freeze-drying and irradiation treatment of collagen all result in the inability to form a gel film. However, using the preparation processes of Examples 1 and 2 of this application, which involve phosphate treatment, freeze-drying, and reverse osmosis treatment of collagen, or further irradiation treatment after freeze-drying of collagen, can form artificial corneas with good light transmittance.

[0118] Furthermore, comparing Examples 1, 2, and Comparative Example 8 (Comparative Example 8 involved mixing dialysis-desalted collagen stock solution with hydrogen peroxide; the collagen did not undergo the pretreatment specified in this application, and no chitosan solution was added to the electrolyte), the results showed that a large number of bubbles were generated during the electrochemical reaction, and no gel film was deposited on the surface of the cathode. This demonstrates that the collagen pretreatment and chitosan addition methods specified in this application are beneficial for obtaining artificial corneas with better light transmittance.

[0119] By comparing Example 1 and Example 13, where Example 13 is a cross-linked artificial cornea and Example 1 is not cross-linked, the light transmittance of the artificial corneas in Example 13 and Example 1 is not significantly different. This indicates that cross-linking artificial corneas with EDC-NHS cross-linking agent does not substantially affect their light transmittance.

[0120] In their experiments, the inventors of this application discovered that the artificial corneas prepared in Examples 8, 11, and 12 were transparent, but exhibited slight collapse after being removed from the cathode surface, and were more prone to cracking during removal. This indicates that the concentration of collagen and chitosan in the electrolyte affects the mechanical properties of the artificial cornea; when the concentration of collagen and / or chitosan in the electrolyte is low, the artificial cornea may become soft or brittle.

[0121] Experimental Example 2

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

[0123] Table 2. Mechanical property 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] As can be seen from the results in Table 2, the tensile strength of the artificial corneas prepared in Examples 1 and 2 of this application is greater than that of the artificial cornea in Comparative Example 5, indicating that the addition of chitosan can improve the tensile strength of the artificial cornea. Furthermore, the artificial cornea prepared in Example 1 of this application exhibits better suture strength.

[0126] The above description is merely a specific embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A method for preparing an artificial cornea, characterized in that, Includes the following steps: An acidic electrolyte is provided, the acidic electrolyte containing collagen, chitosan, and hydrogen peroxide. The collagen in the acidic electrolyte is pretreated collagen. The pretreatment includes a first treatment and a second treatment. The first treatment includes lyophilizing the collagen solution, the collagen solution containing a phosphate solution, and the collagen dissolving in the phosphate solution. The first treatment further includes irradiating the lyophilized collagen. The second treatment includes reverse osmosis treatment of the collagen after the first treatment using an acidic solution. The cathode 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 are inserted into the acidic electrolyte and a power source is applied to carry out 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 steps of treating the collagen after the first treatment with an acid solution by reverse osmosis include: mixing the collagen after the first treatment with water and putting it into a dialysis bag, and then placing the dialysis bag in an acid solution dialysis fluid for 1 to 5 days.

3. The method for preparing an artificial cornea according to claim 2, characterized in that, The concentration of the acid solution is 0.0005~0.02 mol / L.

4. The method for preparing an artificial cornea according to claim 2, characterized in that, The acid solution includes an acetic acid solution.

5. 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.

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

7. The method for preparing an artificial cornea according to claim 1, characterized in that, The preparation steps of the acidic electrolyte include: mixing the collagen after the second treatment with a chitosan solution, adjusting the pH, and then mixing it with a hydrogen peroxide solution to obtain the acidic electrolyte.

8. The method for preparing an artificial cornea according to claim 7, characterized in that, The concentration of collagen in the acidic electrolyte is 0.1~20 mg / mL, and the concentration of chitosan is 0.5~10 mg / mL.

9. The method for preparing an artificial cornea according to any one of claims 1 to 8, characterized in that, The phosphate is composed of monohydrogen phosphate and dihydrogen phosphate.

10. The method for preparing an artificial cornea according to claim 9, characterized in that, The pH value of the phosphate solution is 6-8.

11. The method for preparing an artificial cornea according to claim 9, characterized in that, The preparation steps of the collagen solution include: mixing the collagen stock solution after dialysis and desalting with a phosphate solution to dissolve the collagen in the phosphate solution, thereby obtaining the collagen solution.

12. The method for preparing an artificial cornea according to any one of claims 1 to 8, characterized in that, It also includes crosslinking the gel membrane.

13. The method for preparing an artificial cornea according to claim 1, characterized in that, The irradiation intensity of the irradiation treatment is 5~30KGy.

14. An artificial cornea, characterized in that, It is prepared by the method of any one of claims 1 to 13.

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