A collagen-based artificial cornea with a double-layer structure capable of rapid epithelialization and a preparation method thereof

Abstract

The present invention discloses a collagen-based artificial cornea with a double-layer structure and the ability to rapidly epithelialize, and a preparation method thereof, comprising the following steps: adding collagen to an acetic acid solution, stirring and dissolving, and adding a plasticizer to obtain a high-concentration collagen solution and a low-concentration collagen solution; electrochemically depositing the high-concentration collagen solution to obtain a first collagen film; photocrosslinking the first collagen film by irradiating the first collagen film with strong ultraviolet light; chemically crosslinking the first collagen film to obtain a compacting layer; pouring the low-concentration collagen solution into a sedimentation tank, immersing the compacting layer, and performing a second crosslinking process. The second electrochemical deposition method obtains a second collagen membrane on the compacting layer; the second collagen membrane is subjected to secondary photocrosslinking by weak ultraviolet irradiation to obtain a loose layer, and the loose layer and the compacting layer are combined to form the double-layer structure and the collagen-based artificial cornea capable of rapid epithelialization. The double-layer structure and the collagen-based artificial cornea capable of rapid epithelialization of the present invention have high light transmittance and mechanical properties that meet clinical requirements: the transmittance at 600nm is greater than 90%, the tensile strength is greater than 1Mpa, and the elastic modulus is greater than 10MPa. The low swelling rate can avoid the product from pressuring the autologous corneal tissue, reduce mechanical stimulation to the eye tissue, and improve comfort after implantation.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and in particular to a collagen-based artificial cornea with a double-layer structure capable of rapid epithelialization and a preparation method thereof. Background Art

[0002] The cornea is crucial to the eye's optical function. However, as the outer barrier of the eye, the cornea is susceptible to physical and chemical damage or invasion by pathogens, which can cause corneal diseases and, in severe cases, blindness. For example, corneal opacity caused by mechanical trauma, corneal damage and lesions caused by chemical burns, and corneal scarring caused by bacterial and fungal infections of microbial origin can all lead to a dramatic decrease in vision. Clinically, the most common treatment for severe corneal surface damage is to remove a portion of the patient's diseased lamellar corneal tissue and replace it with a donor cornea. This is called lamellar keratoplasty, which is currently the gold standard for clinical surgery. In traditional lamellar keratoplasty, the depth and diameter of the corneal tissue to be dissected are determined based on the size of the lesion. The lesion is then removed using a trephine drill and an ophthalmic scalpel. Finally, the donor cornea is sutured and fixed to the ocular surface with ophthalmic surgical sutures.

[0003] However, the success of a transplant depends crucially on two factors. First, the rate of epithelialization. Immediately after transplantation, the donor cornea lacks an epithelial cell layer. Limbal stem cells proliferate and migrate, causing corneal epithelial cells to migrate from the edge of the surgical area toward the surgical site, eventually covering the entire donor cornea and forming a multilayered cell layer. This process is called corneal epithelialization. Corneal epithelialization is crucial for postoperative recovery and is a sign of the recipient's acceptance of the donor. The epithelial cell layer serves as the eye's first barrier against the external environment and is crucial for the eye's refractive ability. Rapid epithelialization prevents toxins and microorganisms from infecting the ocular surface. It also protects the transplanted material from damage and degradation caused by eyelid friction and ocular secretions. Therefore, failure to complete epithelialization long after surgery can easily lead to surgical failure. Second, the biocompatibility of the donor cornea itself, as well as its optical and mechanical properties, such as refractive power, transparency, tensile strength, and elastic modulus, are crucial.

[0004] Although lamellar keratoplasty can achieve good results in treating corneal diseases, the shortage of allogeneic donor corneas, which are the gold standard, remains a global obstacle. Therefore, the development of artificial corneas has become an urgent need. At present, artificial corneas used for lamellar transplantation are mainly divided into two categories: acellular biological corneas and biosynthetic artificial corneas. Acellular biological corneas have good biocompatibility and can retain the natural extracellular matrix structure and active substances of the cornea to achieve rapid epithelialization. However, they are mainly derived from porcine corneas. Such animal-derived materials will inevitably have risks such as antigen and virus residues during processing. After implantation, the cornea will become turbid due to a strong rejection reaction. The storage conditions are also relatively harsh and cannot be customized. Before use, doctors need to spend a long time to trim the implant to match the patient's lesions, which prolongs the entire operation time. Biosynthetic artificial corneas have received widespread attention in recent years. Most of them use collagen / recombinant collagen as the base material, and then undergo subsequent performance enhancement designs. They have high biocompatibility, little stimulation to tissue cells, are degradable in the body, have low foreign body sensation, and can imitate the structure and function of the natural cornea to the greatest extent. At the same time, they have the two major advantages of synthetic polymer artificial corneas and decellularized biological corneas, and have potential application value in corneal damage repair.

[0005] At the same time, there are some problems that need to be improved in the biosynthetic artificial cornea. The most commonly used materials currently include collagen, silk fibroin, gelatin, chitosan, etc. These biosynthetic artificial corneas have good biocompatibility and non-toxicity. However, since the current biosynthetic cornea is usually a homogeneous body, the surface hardness of the artificial cornea is relatively high and there are no bioactive substances that promote epithelialization. It is usually impossible to complete epithelialization quickly within 1-2 weeks after surgery. Patent CN118557801 A adds a carboxylated silk fibroin solution dropwise to a collagen solution for cross-linking. After the obtained collagen-carboxylated silk fibroin composite system is cast into a mold, it is fully dried to obtain a carboxylated silk fibroin reinforced collagen-based corneal repair material, but the resulting cornea has an irregular morphology and cannot quickly complete the rapid epithelialization process. Patent CN114618016 A obtains a collagen-based artificial cornea that is consistent with the shape of the cornea on the electrode by extracting collagen, assembling collagen using electrochemical deposition technology, and chemical cross-linking steps. While the cornea produced by this method has a transparency of over 80% and a controllable thickness, its epithelialization process is slow, and the wound cannot close quickly. This dramatically increases the risk of eye infection and can even cause a series of malignant lesions such as corneal ulcers, thereby affecting corneal transparency and scarring, causing irreversible damage to vision. Therefore, developing a biosynthetic artificial cornea that can rapidly epithelialize and isolate the main body of the artificial cornea from the external environment is of great clinical significance for the stable existence and transparency of the biosynthetic artificial cornea in the body. Summary of the Invention

[0006] The purpose of the present invention is to provide a collagen-based artificial cornea with a double-layer structure and capable of rapid epithelialization and a preparation method thereof, so as to overcome the deficiencies in the prior art.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] The present application discloses a method for preparing a collagen-based artificial cornea with a double-layer structure capable of rapid epithelialization, comprising the following steps:

[0009] S1. Preparation of collagen solution: Add collagen to acetic acid solution and stir to dissolve. After complete dissolution, add plasticizer, stir evenly, and centrifuge to degas. Obtain a high-concentration collagen solution and a low-concentration collagen solution. The concentration of the high-concentration collagen solution is 0.3% to 0.5% w / v, and the concentration of the low-concentration collagen solution is 0.1% to 0.25% w / v.

[0010] S2. Primary electrochemical deposition: A high-concentration collagen solution was electrochemically deposited with a platinum mesh connected to the positive electrode, a titanium sheet connected to the negative electrode, and a DC power supply connected to the electrode plate to obtain a collagen film.

[0011] S3. Primary UV crosslinking: After the collagen membrane is dehydrated with anhydrous ethanol, it is photocrosslinked by irradiation with strong UV light;

[0012] S4 chemical cross-linking: The collagen membrane after a UV cross-linking treatment in step S3 was immersed in a 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride solution to obtain a compact layer by chemical cross-linking;

[0013] S5. Secondary electrochemical deposition: The titanium sheet with the compacted layer was connected to the negative electrode, and the platinum mesh was connected to the positive electrode. A low-concentration collagen solution was poured into the deposition tank to immerse the compacted layer, and a secondary electrochemical deposition was performed to obtain a second collagen film on the compacted layer.

[0014] S6. Secondary UV cross-linking: After the collagen membrane 2 is dehydrated with anhydrous ethanol, it is irradiated with weak UV light for secondary photo-cross-linking to obtain a loose layer. The loose layer is combined with the compact layer to form the double-layer structure and the collagen-based artificial cornea capable of rapid epithelialization.

[0015] Preferably, the collagen is type I collagen.

[0016] Preferably, the plasticizer is selected from any one or a combination of glycerol, polyethylene glycol, polypropylene glycol, and citrate plasticizers, and the plasticizer concentration is 10% to 20% v / v.

[0017] Preferably, the concentration of the acetic acid solution is 1% to 3% v / v.

[0018] Preferably, in step S2, the DC power supply selects a constant current mode of 1-4A, and the deposition time is 30-60min; in step S4, the DC power supply selects a constant current mode of 1-4A, and the deposition time is 10-30min.

[0019] Preferably, the intensity of the strong ultraviolet light in step S3 is 600-1000 μw / cm 2 , time is 30~60min; the intensity of weak ultraviolet light in step S6 is 10~200μw / cm 2 , time is 1~10min.

[0020] Preferably, in step S4, the concentration of the 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride solution is 0.01% to 0.04% w / w, and the soaking time is 1 to 5 hours.

[0021] Preferably, the method further includes step S7: washing the double-layered collagen-based artificial cornea capable of rapid epithelialization prepared in step S6 with PBS and storing it in a PBS buffer solution containing recombinant human epidermal growth factor; the concentration of the recombinant human epidermal growth factor is 0.01%-0.03% w / v.

[0022] The present invention also discloses a collagen-based artificial cornea with a double-layer structure capable of rapid epithelialization, which is prepared using the above-mentioned method for preparing a collagen-based artificial cornea with a double-layer structure capable of rapid epithelialization; the invention is applied to corneal diseases caused by corneal damage and corneal infection, and the double-layer artificial cornea of the invention is transplanted into corneal disease patients through lamellar transplantation. The epithelial healing condition of lamellar transplantation affects the recovery of postoperative vision, and even delayed epithelial healing may cause problems such as graft infection, graft matrix dissolution, and ocular surface ulcers. The upper layer of the double-layer structure of the present invention is a loose layer that is easy to degrade, which is conducive to epithelial cell migration and accelerates the lamellar transplantation to complete autologous corneal epithelialization. At the same time, the degradation rate of the lower compact layer is slow, providing a certain thickness support and light penetration performance.

[0023] Beneficial effects of the present invention:

[0024] The present invention provides a bilayer collagen-based artificial cornea capable of rapid epithelialization, with high light transmittance and clinically acceptable mechanical properties: transmittance exceeding 90% at 600 nm, tensile strength exceeding 1 MPa, and elastic modulus exceeding 10 MPa. Its low swelling rate prevents compression of the native corneal tissue, reduces mechanical irritation to ocular tissue, and improves post-implantation comfort.

[0025] 2. Excellent biocompatibility: Made of type I collagen and a small amount of pharmaceutical-grade plasticizer, the artificial cornea is minimally irritating to tissue cells and does not produce an inflammatory response. The artificial cornea has a two-layer structure. The upper layer is a loose layer that is easily degraded, providing an area for epithelial cells to migrate and proliferate. After degradation, it breaks down into small-molecule amino acids, providing nutrients for tissue cells. This also facilitates rapid epithelialization during lamellar transplantation surgery, preventing the lesion from contacting the outside world and potentially causing infection.

[0026] 3. Long-term stability and synergistic promotion of epithelialization: The lower layer is a compacting layer with a slower degradation rate, providing a certain thickness support for the cornea and cooperating with other corneal structures to achieve refractive function. Furthermore, by increasing cross-linking strength, it can maintain long-term stability. The epidermal growth factor carried on the surface of the artificial cornea can synergistically promote the rapid growth of corneal epithelial cells, complete epithelialization, and ensure that the main body of the artificial cornea material is isolated from the external environment, preventing adverse effects such as degradation, infection, and inflammation on the material and surrounding tissues. The simple operation steps facilitate large-scale production.

[0027] The features and advantages of the present invention will be described in detail through embodiments with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the proliferation of cells in Example 2.

[0029] Figure 2 Schematic diagram of the penetration experiment.

[0030] Figure 3 This is a trend chart of in vitro enzymatic hydrolysis.

[0031] Figure 4 The figure shows the degradation trend in vitro simulated using artificial tears.

[0032] Figure 5 Schematic diagram of an artificial cornea with a collagen-based double-layer structure.

[0033] Figure 6 Fluorescent staining of the rabbit epithelium on the second day and one week after lamellar corneal transplantation. DETAILED DESCRIPTION

[0034] To make the objectives, technical solutions, and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and examples. However, it should be understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the scope of the present invention. In addition, in the following description, descriptions of well-known structures and technologies are omitted to avoid unnecessary confusion of the present invention.

[0035] The present invention provides a method for preparing a collagen-based artificial cornea with a double-layer structure capable of rapid epithelialization, comprising the following steps:

[0036] S1. Preparation of collagen solution: Add collagen to acetic acid solution and stir to dissolve. After complete dissolution, add plasticizer, stir evenly, and centrifuge to degas. Obtain a high-concentration collagen solution and a low-concentration collagen solution. The concentration of the high-concentration collagen solution is 0.3% to 0.5% w / v, and the concentration of the low-concentration collagen solution is 0.1% to 0.25% w / v.

[0037] Specifically, the collagen is type I collagen; the plasticizer is selected from any one or a combination of glycerol, polyethylene glycol, polypropylene glycol, and citrate plasticizers, and the plasticizer concentration is 10% to 20% v / v; the acetic acid solution concentration is 1% to 3% v / v

[0038] S2. Primary electrochemical deposition: A high-concentration collagen solution was electrochemically deposited with a platinum mesh connected to the positive electrode, a titanium sheet connected to the negative electrode, and a DC power supply connected to the electrode plate to obtain a collagen film.

[0039] Specifically, the DC power supply selected the constant current mode of 1~4A, and the deposition time was 30~60min;

[0040] S3. Primary UV crosslinking: After the collagen membrane is dehydrated with anhydrous ethanol, it is photocrosslinked by irradiation with strong UV light;

[0041] Specifically, the intensity of strong ultraviolet light is 600~1000μw / cm 2 , time is 30~60min;

[0042] S4 chemical cross-linking: The collagen membrane after a UV cross-linking treatment in step S3 was immersed in a 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride solution to obtain a compact layer by chemical cross-linking;

[0043] Specifically, the concentration of the 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride solution is 0.01% to 0.04% w / w, and the soaking time is 1 to 5 hours;

[0044] S5. Secondary electrochemical deposition: The titanium sheet with the compacted layer was connected to the negative electrode, and the platinum mesh was connected to the positive electrode. A low-concentration collagen solution was poured into the deposition tank to immerse the compacted layer, and a secondary electrochemical deposition was performed to obtain a second collagen film on the compacted layer.

[0045] Specifically, the DC power supply selects a constant current mode of 1~4A, and the deposition time is 10~30min.

[0046] S6. Secondary UV crosslinking: After the collagen membrane is dehydrated with anhydrous ethanol, it is irradiated with weak UV light for secondary photocrosslinking to obtain a loose layer. The loose layer is combined with the compact layer to form the double-layer structure and the collagen-based artificial cornea capable of rapid epithelialization. Figure 5 .

[0047] Among them, the intensity of weak ultraviolet light is 10~200μw / cm 2 , time is 1~10min.

[0048] The method further includes step S7: washing the double-layered collagen-based artificial cornea capable of rapid epithelialization prepared in step S6 with PBS and storing it in a PBS buffer solution containing recombinant human epidermal growth factor; the concentration of the recombinant human epidermal growth factor is 0.01%-0.03% w / v.

[0049] Example 1

[0050] In this embodiment, a method for preparing an artificial cornea with a collagen-based double-layer structure is provided, as follows:

[0051] (1) Preparation of collagen solution: Use 1% v / v acetic acid solution as solvent to prepare 0.3% w / v high-concentration collagen solution and 0.1% w / v low-concentration collagen solution. Stir and dissolve until there is no obvious lumps in the solution. After complete dissolution, add 10% v / v plasticizer and stir evenly.

[0052] (2) Bubble removal: The collagen solution was centrifuged for degassing at a speed of 8000 rpm for 15 min.

[0053] (3) Primary electrochemical deposition: Under a low temperature environment of 4°C, 0.3% w / v high concentration collagen solution was electrochemically deposited. The platinum mesh was connected to the positive electrode, the titanium sheet was connected to the negative electrode, and the electrode plate was connected to a DC source for electrochemical deposition. The collagen film material was obtained on the titanium sheet. The DC power supply selected the constant current mode of 1A, and the deposition time was 30 min.

[0054] (4) Single UV cross-linking: The collagen membrane on the titanium sheet was immersed in anhydrous ethanol for dehydration and irradiated with 600 μw / cm2 UV light for 30 min for UV cross-linking.

[0055] (5) Chemical cross-linking: Soak the collagen membrane in 0.01% w / w DMTMM solution for 1 h to perform chemical cross-linking to obtain a compact layer.

[0056] (6) Secondary electrochemical deposition: Under a low temperature environment of 8°C, the titanium sheet with a compacted layer was connected to the negative electrode, the platinum mesh was connected to the positive electrode, and a 0.1% w / v low-concentration collagen solution was poured into the deposition tank to immerse the compacted layer for secondary electrochemical deposition. The DC power supply was selected in constant current mode of 1A, and the deposition time was 10 min.

[0057] (7) Secondary UV cross-linking: A new layer of collagen membrane is covered on the compacted layer, dehydrated with ethanol, and irradiated under 10 μw / cm2 UV light for 10 min for UV cross-linking.

[0058] (8) Peeling off the membrane: Soak the titanium sheet and collagen membrane in pure water together. When the collagen membrane is fully moistened, slowly peel the collagen membrane off the titanium sheet to obtain a double-layer artificial cornea.

[0059] (9) PBS cleaning: The artificial cornea was immersed in PBS buffer solution and shaken for cleaning. 1L PBS solution was shaken for 10 minutes, and washed 5 times in total.

[0060] (10) Storage method: The prepared artificial cornea was stored in PBS buffer containing 0.01% w / v rhEGF.

[0061] Example 2

[0062] In this embodiment, a method for preparing an artificial cornea with a collagen-based double-layer structure is provided, as follows:

[0063] (1) Preparation of collagen solution: Use 2% v / v acetic acid solution as solvent to prepare 0.38% w / v high-concentration collagen solution and 0.12% w / v low-concentration collagen solution. Stir and dissolve until there is no obvious lumps in the solution. After complete dissolution, add 15% v / v plasticizer and stir evenly.

[0064] (2) Bubble removal: The collagen solution was centrifuged for degassing at a speed of 8000 rpm for 15 min.

[0065] (3) Primary electrochemical deposition: Under a low temperature environment of 5°C, 0.38% w / v high concentration collagen solution was electrochemically deposited. The platinum mesh was connected to the positive electrode, the titanium sheet was connected to the negative electrode, and the electrode plate was connected to a DC source for electrochemical deposition. The collagen film material was obtained on the titanium sheet. The DC power supply selected the constant current mode of 2A, and the deposition time was 40 min.

[0066] (4) Single UV cross-linking: The collagen membrane on the titanium sheet was immersed in anhydrous ethanol for dehydration and irradiated with 800 μw / cm2 UV light for 40 min.

[0067] (5) DMTMM cross-linking: The collagen membrane was soaked in 0.02% w / w DMTMM solution for 4 h to perform chemical cross-linking to obtain a compact layer.

[0068] (6) Secondary electrochemical deposition: At a low temperature of 4°C, the titanium sheet with a compacted layer was connected to the negative electrode, the platinum mesh was connected to the positive electrode, and a 0.12% w / v low-concentration collagen solution was poured into the deposition tank to immerse the compacted layer for secondary electrochemical deposition. The DC power supply was selected in constant current mode of 2A, and the deposition time was 15 min.

[0069] (7) Secondary UV cross-linking: A new layer of collagen membrane is covered on the compacted layer, dehydrated with ethanol, and irradiated under 100 μw / cm2 UV light for 2.5 min for UV cross-linking.

[0070] (8) Peeling off the membrane: Soak the titanium sheet and collagen membrane in pure water together. When the collagen membrane is fully moistened, slowly peel the collagen membrane off the titanium sheet to obtain a double-layer artificial cornea.

[0071] (9) PBS cleaning: The artificial cornea was immersed in PBS buffer solution and shaken for cleaning. 3L PBS solution was shaken for 30 minutes, and a total of 8 cleanings were performed.

[0072] (10) Storage method: The prepared artificial cornea was stored in PBS buffer containing 0.018% w / v rhEGF.

[0073] Example 3

[0074] In this embodiment, a method for preparing an artificial cornea with a collagen-based double-layer structure is provided, as follows:

[0075] (1) Preparation of collagen solution: Use 3% v / v acetic acid solution as solvent to prepare 0.5% w / v high-concentration collagen solution and 0.25% w / v low-concentration collagen solution. Stir and dissolve until there is no obvious lumps in the solution. After complete dissolution, add 20% v / v plasticizer and stir evenly.

[0076] (2) Bubble removal: The collagen solution was centrifuged for degassing at a speed of 8000 rpm for 15 min.

[0077] (3) Primary electrochemical deposition: Under a low temperature environment of 8°C, 0.5% w / v high concentration collagen solution was electrochemically deposited. The platinum mesh was connected to the positive electrode, the titanium sheet was connected to the negative electrode, and the electrode plate was connected to a DC source for electrochemical deposition. The collagen film material was obtained on the titanium sheet. The DC power supply selected the constant current mode of 4A, and the deposition time was 60 min.

[0078] (4) Single UV cross-linking: The collagen membrane on the titanium sheet was immersed in anhydrous ethanol for dehydration and irradiated with 1000 μw / cm2 UV light for 60 min.

[0079] (5) DMTMM cross-linking: The collagen membrane was soaked in 0.04% w / w DMTMM solution for 5 h to perform chemical cross-linking to obtain a compact layer.

[0080] (6) Secondary electrochemical deposition: Under a low temperature environment of 5°C, the titanium sheet with a compacted layer was connected to the negative electrode, the platinum mesh was connected to the positive electrode, and a 0.25% w / v low-concentration collagen solution was poured into the deposition tank to immerse the compacted layer for secondary electrochemical deposition. The DC power supply selected the constant current mode of 4A and the deposition time was 30 min.

[0081] (7) Secondary UV cross-linking: A new layer of collagen membrane is covered on the compacted layer, dehydrated with ethanol, and irradiated under 200 μw / cm2 UV light for 1 min for UV cross-linking.

[0082] (8) Peeling off the membrane: Soak the titanium sheet and collagen membrane in pure water together. When the collagen membrane is fully moistened, slowly peel the collagen membrane off the titanium sheet to obtain a double-layer artificial cornea.

[0083] (9) PBS cleaning: The artificial cornea was immersed in PBS buffer solution and shaken for cleaning. 4L PBS solution was shaken for 60 minutes, and a total of 10 cleanings were performed.

[0084] (10) Storage method: The prepared artificial cornea was stored in PBS buffer containing 0.03% w / v rhEGF.

[0085] Comparative Example 1

[0086] The only difference between this comparative example and Example 2 is that no plasticizer is added to the collagen solution.

[0087] Comparative Example 2

[0088] The only difference between this comparative example and Example 2 is that ultraviolet light is not used to photocrosslink the artificial cornea.

[0089] Comparative Example 3

[0090] The only difference between this comparative example and Example 2 is that DMTMM is not used to chemically cross-link the artificial cornea.

[0091] Comparative Example 4

[0092] The only difference between this comparative example and Example 2 is that no secondary electrochemical deposition is performed.

[0093] Experiment 1

[0094] The artificial corneas obtained in the above examples and comparative examples were tested for light transmittance, water content, mechanical properties, swelling rate, and ion permeability. Five parallel samples were tested in each group, and the average value was taken. The specific testing methods are as follows:

[0095] 1. Transmittance: Take a sample, wipe off the surface moisture with absorbent paper, place the sample in the center of the transmittance meter, and select the 600nm band for measurement.

[0096] 2. Moisture content: Take a sample and determine the moisture content according to the second method, drying method, in Part IV, 0832, Determination of Moisture in the Pharmacopoeia (2020 edition).

[0097] 3. Mechanical properties: Take the specially prepared sample, measure its width and thickness, and test it at room temperature at a tensile rate of 50 mm / min.

[0098] 4. Swelling rate: Weigh the sample and record the mass. Place the sample in a swelling tube, add 20 mL of solvent, cap the tube tightly, and place it in a thermostatic bath at 25°C ± 0.1°C. Weigh the mass of the swelling body every 3 hours using the same method until the difference between the two weighing results does not exceed 0.019, at which point the swelling equilibrium is considered to have been reached.

[0099] 5. Osmotic performance: The sample is fixed between the osmotic chamber (containing nutrient solution) and the receptor chamber (containing deionized water), and then the solutions in the two chambers are uniformly stirred with an electromagnetic stirrer. The concentration of the nutrient solution (tryptophan solution) in the receptor chamber is then tested. The principle of calculating the permeability coefficient is as shown in the attached Figure 2 Shown:

[0100] where P is the permeability coefficient; V and S represent the volume of the solution in the chamber and the membrane area between the chambers, respectively; d represents the thickness of the wet sample; t is the diffusion time; C0 is the initial ion concentration in the permeation chamber, and C is the ion concentration in the receptor chamber at the target time (1, 6, 12, 18, and 24 h of immersion, respectively).

[0101] 6. In vitro enzymatic hydrolysis performance: Under the clean bench, measure 5mL of Tris-HCl solution and put it into a 15mL centrifuge tube. Use tweezers to pick up the artificial cornea, put it into a sterile centrifuge tube containing 5mL of Tris-HCl solution, seal it, and place it in a 37°C water bath for 1 hour. After the end, take out the artificial cornea clamp, gently wipe off the surface moisture, and weigh it as the original weight. Then put the mother liquor activated in a 37°C water bath for 1 hour into the above centrifuge tube, with 88.3μL of mother liquor in each tube, shake well, seal it, and place it at 37°C for in vitro enzymatic hydrolysis experiment observation. Change the enzyme solution every 8 hours (it needs to be activated for 1 hour before use) to maintain enzyme activity. Observe and record the status and residual weight of the sample until the sample is completely enzymatically hydrolyzed and the degradation trend is as shown in the attached figure. Figure 3 shown.

[0102] 7. Artificial tear simulation in vitro degradation: Weigh the artificial cornea as the initial weight. Under the clean bench, measure 5mL of artificial tears and place it in a 15mL centrifuge tube. Use tweezers to pick up the artificial cornea and place it in the above sterile centrifuge tube. Seal it and place it in a 37℃ constant temperature shaker. Observe and record the state of the sample and the remaining weight until the sample is completely degraded and the degradation trend is as shown in the attached figure. Figure 4 shown.

[0103] The above test results are shown in Table 1 below.

[0104] Table 1

[0105]

[0106] The results showed that the artificial cornea prepared by the method of the present invention had a transmittance of more than 90% at 600nm, excellent optical properties, and avoided the occurrence of light blocking caused by the sample itself. The water content was more than 85%, and the transmittance and water content were similar to those of a healthy human cornea. The tensile strength was above 1.5MPa, the elastic modulus was higher than 10MPa, and the mechanical properties were excellent. The swelling rates of the artificial corneas in Examples 1-3 were all lower than 15%, which could avoid nerve compression of the autologous cornea by the product, reduce mechanical stimulation of the eye tissue, and improve comfort after implantation. At the same time, the tryptophan permeability coefficient was similar to that of a healthy human cornea, proving that this product can support the transmission of small molecule nutrients, ensure the absorption of nutrients by surrounding tissues after surgery, and promote wound repair. In terms of the degradation time of artificial tears simulated in vitro, the period was about 80-100 days. As time increased, the upper layer of the artificial cornea began to degrade after one week, while the lower layer remained intact. The in vitro enzymatic hydrolysis time for the examples was over 90 hours, while the enzymatic resistance of the comparative example was significantly weakened, indicating that the cross-linking method of this product significantly improves its resistance to enzymatic hydrolysis. The upper layer of the bilayer structure, the loose layer, degraded faster than the compact layer under both simulated degradation conditions, facilitating epithelial cell migration and proliferation. The collagen in the loose layer degrades into small molecule amino acids, providing the cells with the nutrients they need, promoting epithelialization, ensuring rapid sealing and closure of the corneal tissue, and preventing external bacteria from entering the corneal tissue and causing subsequent infection. The lower layer, the compact layer, degrades more slowly, acting as a guide for tissue regeneration, providing a certain thickness support and light transmission properties for the cornea.

[0107] Experiment 2

[0108] Cytotoxicity test: Under sterile operation, the cornea in Example 2 was taken and the 215.00 mL of extraction medium (MEM with 10% fetal bovine serum (1xMEM)) was added at a ratio of 10% to 10% of the total serum. The extracted solution was obtained after extraction at 37°C for 24 h. The extracted solution was filtered with a 0.22 μm sterile filter membrane before use. The cell suspension with a density of 1 × 104 cells / mL was inoculated into each set of parallel wells. 100 mL of cell suspension was inoculated into each well. The solution was incubated at 37°C with 5% CO 2 Cultured in an incubator for 24 hours, discarded the original culture medium after the end of the culture, added sample extract, each well in 100mL. Then continue to culture for 72 hours, after the end of the culture, observe the morphology of the cultured cells under a microscope, the results are shown in the attached Figure 1 .

[0109] The results show that the artificial cornea prepared by the method of the present invention has good cell affinity and biocompatibility, has little stimulation to cells, and can ensure the normal growth of cells.

[0110] Experiment 3

[0111] Observation of epithelialization in rabbit corneal lamellar transplantation surgery: New Zealand rabbits weighing 2.5 to 3 kg were selected for in vivo animal experiments. After general anesthesia, local anesthesia was performed using oxybuprocaine hydrochloride eye drops, and the surgery was performed under a sterile environment. A 4.5 mm trephine drill was used to drill a 150-200 μm deep implant bed in the center of the rabbit cornea. During the surgery, the cornea in Example 2 and the cornea in Comparative Example 4 were taken out of the preservation solution and sutured with 10-0 surgical sutures, respectively. On the second day and the first week after surgery, fluorescein sodium ophthalmic test paper was moistened at the implantation site to perform fluorescent staining on the defective area, and cobalt blue slit lamp fluorescent staining photography was used to observe the corneal epithelialization. Figure 6 .

[0112] Experimental results show that the double-layered artificial cornea produced by the method described in this invention has a faster epithelialization rate. This is due to the fact that clinical trials have shown that the release of rhEGF significantly promotes the healing of corneal epithelial damage. Furthermore, the thinner and looser upper layer of the implant facilitates cell adhesion and promotes epithelial cell migration, thereby accelerating the epithelialization repair process. Other biosynthetic artificial corneas, designed with only a single, compact surface, have a smooth surface that is not conducive to cell climbing, resulting in a slower epithelialization rate and ultimately unsatisfactory clinical results.

[0113] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing a collagen-based artificial cornea with a double-layer structure and capable of rapid epithelialization, characterized in that: The steps include: S1. Preparation of collagen solution: Add collagen to acetic acid solution and stir to dissolve. After complete dissolution, add plasticizer, stir evenly, and centrifuge to degas. Obtain a high-concentration collagen solution and a low-concentration collagen solution. The concentration of the high-concentration collagen solution is 0.3% to 0.5% w / v, and the concentration of the low-concentration collagen solution is 0.1% to 0.25% w / v. S2. Primary electrochemical deposition: A high-concentration collagen solution was electrochemically deposited with a platinum mesh connected to the positive electrode, a titanium sheet connected to the negative electrode, and a DC power supply connected to the electrode plate to obtain a collagen film. S3. Primary UV crosslinking: After the collagen membrane is dehydrated with anhydrous ethanol, it is photocrosslinked by irradiation with strong UV light; S4 chemical cross-linking: The collagen membrane after a UV cross-linking treatment in step S3 was immersed in a 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride solution to obtain a compact layer by chemical cross-linking; S5. Secondary electrochemical deposition: The titanium sheet with the compacted layer was connected to the negative electrode, and the platinum mesh was connected to the positive electrode. A low-concentration collagen solution was poured into the deposition tank to immerse the compacted layer, and a secondary electrochemical deposition was performed to obtain a second collagen film on the compacted layer. S6. Secondary UV crosslinking: After the collagen membrane is dehydrated with anhydrous ethanol, it is irradiated with weak UV light for secondary photocrosslinking to obtain a loose layer. The loose layer is combined with the compact layer to form the double-layer structure and the collagen-based artificial cornea capable of rapid epithelialization. The intensity of the strong UV light in step S3 is 600-1000 μw / cm 2 , time is 30~60min; the intensity of weak ultraviolet light in step S6 is 10~200μw / cm 2 , time is 1~10min.

2. The method for preparing a collagen-based artificial cornea with a double-layer structure capable of rapid epithelialization according to claim 1, characterized in that: The collagen is type I collagen.

3. The method for preparing a collagen-based artificial cornea with a double-layer structure capable of rapid epithelialization according to claim 1, characterized in that: The plasticizer is selected from any one or a combination of glycerol, polyethylene glycol, polypropylene glycol, and citrate plasticizers, and the concentration of the plasticizer is 10% to 20% v / v.

4. The method for preparing a collagen-based artificial cornea with a double-layer structure capable of rapid epithelialization according to claim 1, characterized in that: The concentration of the acetic acid solution is 1% to 3% v / v.

5. The method for preparing a collagen-based artificial cornea with a double-layer structure capable of rapid epithelialization according to claim 1, characterized in that: In step S2, the DC power supply selects a constant current mode of 1-4A, and the deposition time is 30-60 minutes; in step S5, the DC power supply selects a constant current mode of 1-4A, and the deposition time is 10-30 minutes.

6. The method for preparing a collagen-based artificial cornea with a double-layer structure capable of rapid epithelialization according to claim 1, characterized in that: In step S4, the concentration of the 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride solution is 0.01% to 0.04% w / w, and the soaking time is 1 to 5 hours.

7. The method for preparing a collagen-based artificial cornea with a double-layer structure capable of rapid epithelialization according to claim 1, characterized in that: The method further includes step S7: washing the double-layered collagen-based artificial cornea capable of rapid epithelialization prepared in step S6 with PBS and storing it in a PBS buffer solution containing recombinant human epidermal growth factor; the concentration of the recombinant human epidermal growth factor is 0.01%-0.03% w / v.

8. A collagen-based artificial cornea with a double-layer structure capable of rapid epithelialization, characterized by: The invention discloses a collagen-based artificial cornea having a double-layer structure and capable of rapid epithelialization, prepared by the method for preparing the collagen-based artificial cornea having a double-layer structure and capable of rapid epithelialization as described in any one of claims 1 to 7; the collagen-based artificial cornea having a double-layer structure and capable of rapid epithelialization is used for corneal diseases requiring lamellar transplantation surgery caused by corneal injury or corneal infection.

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