Collagen corneal graft with bionic structure and preparation method of collagen corneal graft

Abstract

The invention provides a structure bionic collagen corneal graft and a preparation method thereof. The preparation method of the collagen corneal graft with the bionic structure comprises the following steps: S1, dissolving collagen to prepare a collagen solution; s2, adding a cross-linking agent into the collagen solution to carry out a cross-linking reaction; and S3, carrying out bubble removal, molding, freeze thawing, freeze drying, washing and drying on the cross-linking reaction product to prepare the structure bionic collagen corneal graft. The water content of the collagen cornea graft with the bionic structure is similar to that of a natural cornea, the light transmittance performance is good, the light transmittance and a light transmittance curve in a visible light range are not greatly different from those of tissues, the ion permeability performance and the biomechanical performance are good, and the requirements of nutrient substance transportation and eyeball supporting and protecting effects can be met; particularly, the cornea graft has excellent suture resistance, can bear suture and tearing of operation lines, and has a good cornea repair effect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of corneal repair, in particular to a structure-bionic collagen corneal graft and a preparation method thereof. BACKGROUND

[0002] The human cornea is a transparent tissue with unique dioptric, nutritional permeability and biomechanical properties, and has physiological functions such as participating in dioptric, maintaining intraocular pressure and protecting eyeball. The corneal stroma accounts for more than 90% of the thickness of the cornea, and is mainly composed of collagen fibers arranged in regular lamellas. If a biomaterial is to replace the physiological functions of the natural cornea, it is necessary to simulate its structure and function. However, the commercially available Boston keratoprosthesis, bone-tooth type keratoprosthesis, AlphaCor keratoprosthesis and KPro keratoprosthesis have postoperative complications such as easy formation of posterior capsule, endophthalmitis, corneal stromal dissolution or intraocular pressure rise after implantation. Therefore, it is expected to find a biomaterial graft that can better integrate into the patient's eye and possibly trigger the self-regeneration process of damaged tissue.

[0003] At present, the research of biomaterial graft mainly includes the following categories: one category is the synthetic non-regenerative keratoprosthesis using PMMA, PHEMA, PVA and other substrates, which has the disadvantages of non-degradable, unable to biologically heal, many surgical complications and the like; another category is the porcine corneal acellular matrix, which has the disadvantages of complex composition, rejection risk, and inability to customize curvature. In addition, domestic and foreign scholars use physical, chemical and biological methods to modify natural polymer materials or degradable synthetic polymer materials to prepare various corneal repair materials. The modified corneal repair materials have improved transparency, stability and mechanical properties, but still have various problems such as poor mechanical strength and toughness, inability to be sutured and fixed, poor transparency, etc., making it difficult to achieve the reconstruction of corneal function and the practical application of corneal repair materials in clinic.

[0004] In view of this, the present application is proposed. SUMMARY

[0005] The purpose of the present application is to provide a structure-bionic collagen corneal graft and a preparation method thereof, which has excellent biocompatibility, transparency, ion permeability and biomechanical properties, can withstand suture and tear of surgical thread, and has good corneal repair effect.

[0006] The present application provides a preparation method of a structure-bionic collagen corneal graft, comprising the following steps:

[0007] S1: dissolving collagen to obtain a collagen solution;

[0008] S2: adding a crosslinking agent to the collagen solution for crosslinking reaction;

[0009] S3: degassing, molding, freeze-thawing, freeze-drying, washing, drying the cross-linking reaction product to obtain a structure biomimetic collagen corneal implant (referred to as a corneal implant).

[0010] In step S1, the collagen is type I collagen extracted from bovine Achilles tendon, which can be purchased from Guangzhou Pudao Lianxin Biological Technology Co., Ltd.

[0011] The collagen is dissolved in a hydrochloric acid solution; the concentration of the hydrochloric acid solution can be 0.005-0.015 mol / L, and the pH value of the hydrochloric acid solution can be 1.5-2.5; the ratio of the amount of the hydrochloric acid solution to the amount of the collagen can be (80-120) mL:(10-50) mg; the dissolution time can be 10-16 h; and the concentration of the collagen solution can be 0.1-0.5 mg / mL.

[0012] In step S2, the cross-linking agent is at least one selected from carbodiimide, genipin and citric acid. Currently, the conventional cross-linking agent usually adopts the following two kinds: 1) riboflavin, which is mainly molded by light curing, but compared with the human natural cornea, it may still be insufficient, and the long-term stability and anti-degradation ability are poor, and there may be a risk of deformation or rupture in the mechanical environment of the eye; in addition, the cross-linking process may introduce incompletely reacted riboflavin or by-products, which is easy to cause inflammatory reaction, foreign body reaction and even graft rejection; 2) glutaraldehyde, etc., which mainly forms a covalent cross-linking through Schiff base reaction between the aldehyde groups at both ends of the molecule and the free amino groups on the collagen molecule, but it has significant cytotoxicity, and the residual aldehyde group or unreacted glutaraldehyde will cause strong inflammatory reaction and adverse reactions of the tissue at the implant site, which seriously limits its application in contact with living cells; in addition, the cross-linked collagen material is often excessively rigid and increases in brittleness, lacks the flexibility and elasticity of natural tissue, and may lead to mismatch of mechanical properties. However, the present application uses carbodiimide, genipin and citric acid as cross-linking agents, which are different from the above-mentioned light-cured cross-linking agents and conventional cross-linking agents such as glutaraldehyde. The above-mentioned specific cross-linking agents of the present application mainly form natural amide bonds between the carboxyl and amino groups on the collagen molecule through catalysis, which has the advantages of very low cytotoxicity, good cell affinity, significantly improved material toughness, etc.

[0013] More specifically, the cross-linking agent is added in the form of a cross-linking agent solution; the mass concentration of the cross-linking agent solution can be 3-10%, and the volume ratio of the collagen solution to the cross-linking agent solution can be 100 mL:10-20 mL. In addition, the temperature of the cross-linking reaction can be 1-10℃, for example, 1-4℃; and the time of the cross-linking reaction can be 1-3 h, for example, 1-2 h, at which time the cross-linking agent has not completely reacted with the collagen, and the unreacted cross-linking agent will continue to react in the subsequent freeze-thawing and freeze-drying processes.

[0014] In step S3, the bubble removing includes: stirring the cross-linking reaction product at a rotating speed of 160-200 r / min, and the stirring time can be 3-6 h.

[0015] The present application adopts the female die and the male die with curvature to form, and it can be understood that the female die and the male die match with each other to form a closed cavity for forming the structural biomimetic collagen corneal graft; the curvature radius of the female die and the male die is 9.5-10 mm, and the chord length is 14-15 mm. At present, the curvature radius of the existing artificial cornea is usually 6.5-6.8 mm, which is close to the curvature radius of the human cornea; however, the present inventor finds through experimental research that when the curvature radius is 6.5-6.8 mm, the artificial cornea which is too steep protrudes forward, which causes that the material cannot be well combined with the corneal tissue to form a gap, and thus postoperative infection is easily caused; at the same time, the corneal tissue cannot be completely epithelized due to the poor combination with the implant bed, and thus serious graft dissolution occurs. Through a large number of experimental researches, the present application designs the curvature radius as 9.5-10 mm; the test results show that the relatively flat artificial cornea does not protrude too much forward, and the transition of the curvature with the surrounding recipient corneal tissue is smoother and more natural, which can better maintain the normal anterior chamber depth, and is not easy to cause postoperative infection or graft dissolution and other problems.

[0016] Further, the freeze-thawing includes: first freezing at-70℃ to-90℃ for 50-70 min, and then thawing at 2-6℃ for 20-30 h; the freeze-drying temperature is-15℃ to-25℃, and the freeze-drying time is 45-50 h. Researches show that the specific freeze-thawing and freeze-drying in the forming process can further adjust and stabilize the physical cross-linking of the collagen fiber network through repeated formation and melting of ice crystals; the growth of the ice crystals will expel the collagen molecules, so that they are more closely gathered to form a thicker and more stable fiber structure, which provides a more optimal and initially formed physical basis for the subsequent chemical cross-linking; the unreacted cross-linking agent continues to react in the solid or semi-solid state in the formed network, which can better play the cross-linking role of the cross-linking agent, and endow the material with higher compression modulus, tensile strength, tear resistance and elastic modulus.

[0017] The structural biomimetic collagen corneal graft of the present application has the following structure and performance: the shape of the structural biomimetic collagen corneal graft is a circular arc, the curvature radius is 9.5-10 mm, and the chord length is 14-15 mm; the water content of the structural biomimetic collagen corneal graft can reach more than 93%, the light transmittance in the range of 800 nm visible light can reach 90%, and the tear resistance of 20 g weight can be borne, and the collagenase enzymatic hydrolysis experiment in vitro is completely degraded until 44 h. In addition, after the rabbit corneal lamellar transplantation surgery, the corneal graft is completely epithelized on the 7th day, and is completely fused with the surrounding corneal tissue after 2 weeks, and good transparency is achieved.

[0018] The application also provides a structure-bionic collagen corneal implant prepared according to the preparation method.

[0019] Compared with the prior art, the application has at least the following advantages:

[0020] 1. According to the principle of "component-structure-function" bionic design, the application adopts a new type of biological crosslinking method to construct a structure-bionic collagen corneal implant based on natural collagen, and the structure-bionic collagen corneal implant has excellent biocompatibility, transparency and biomechanical properties.

[0021] 2. The structure-bionic collagen corneal implant has a water content rate similar to that of a natural cornea, good light transmission performance, no great difference between the light transmission rate and the light transmission curve in the visible light range, good ion permeation performance and biomechanical properties, and can meet the transportation of nutrients and the support and protection of eyeballs.

[0022] 3. The structure-bionic collagen corneal implant has excellent suture resistance and can withstand suturing and tearing of surgical threads, and has good corneal repair effect. BRIEF DESCRIPTION OF DRAWINGS

[0023] In order to more clearly illustrate the specific embodiments of the application or the technical solutions in the prior art, the drawings needed in the specific embodiments or the prior art description will be briefly introduced below. Obviously, the drawings in the following description are some embodiments of the application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0024] Figure 1 Structure schematic diagram of the female mold and the male mold used for molding;

[0025] Figure 2 Actual photograph of the structure-bionic collagen corneal implant in a wet state;

[0026] Figure 3 Water content rate detection results of the corneal implants;

[0027] Figure 4 Light transmission rate detection results of the corneal implants;

[0028] Figure 5 Mechanical property detection results of the corneal implants;

[0029] Figure 6 Type I collagenase degradation performance detection results of the corneal implants;

[0030] Figure 7 Weight experiment results of the corneal implant in Example 1;

[0031] Figure 8 The photos of the surgical suturing of the corneal graft of Example 1;

[0032] Figure 9 The animal experiment results of the corneal graft of Example 1; wherein: (A) is the postoperative photo of the lamellar keratoplasty of the corneal regeneration repair material, (B) is the photo of the fluorescein sodium staining for detecting the epithelialization process and slit lamp;

[0033] Figure 10 The repair effect of the corneal graft of Example 1; wherein: (A) is the H&E staining of the healthy corneal section 6 months after the operation, (B) is the H&E staining of the corneal graft section, ep is the epithelial cell layer, s is the stroma layer, en is the endothelial cell layer, and the arrow is the newly born corneal stromal cell;

[0034] Figure 11 The clinical experiment results of the corneal graft of Example 1. DETAILED DESCRIPTION

[0035] It should be noted that the following detailed description is illustrative only and is intended to provide further description of the present application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application pertains.

[0036] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments in accordance with the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and / or "comprising," when used in this specification, specify the presence of stated features, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof.

[0037] The technical solutions of the present application will be described below in conjunction with the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0038] Example 1

[0039] The preparation method of the structure-biomimetic collagen corneal graft of the present embodiment is as follows:

[0040] All experimental instruments and deionized water were sterilized in a high-temperature and high-pressure sterilizer, and then sealed with plastic wrap for standby use.

[0041] A bag of collagen gel (type I collagen extracted from bovine Achilles tendon, from Guangzhou Pudao Lianxin Biotechnology Co., Ltd.) was taken out of a -20℃ freezer and thawed at room temperature for 24 hours. 1g of the collagen gel (10mg collagen content) was torn into small pieces and placed in a 500mL Erlenmeyer flask. 100mL of 0.01mol / L, pH=2 hydrochloric acid solution was added to the flask and dissolved for 10 hours to obtain a collagen solution with a concentration of 0.1mg / mL.

[0042] 10 mL of a 10% (w / w) citric acid crosslinking agent solution was added to the collagen solution via a separatory funnel. The crosslinking reaction was carried out at 1°C for 1 h with stirring to obtain the crosslinking product.

[0043] The crosslinking reaction product was moved to room temperature and stirred at 180 rpm for 4 hours to remove air bubbles; subsequently, it was combined with... Figure 1 As shown, 2 mL of the degassed cross-linking reaction product was dropped into the cavity of a negative mold (radius of curvature 9.9 mm, chord length 14 mm). The matching positive mold was then pressed together with the negative mold to form a sealed cavity containing the cross-linking reaction product. The cavity was then first frozen at -80℃ for 1 hour, then thawed at 4℃ for 24 hours, and finally freeze-dried at -20℃ for 48 hours to produce a collagen material with a curved shape. Residual hydrochloric acid was washed away with deionized water, and the pH of the solution after washing was measured in real-time using a pH meter. Once the pH reached 7.0, the solution was dried in a mold to obtain a biomimetic collagen corneal graft (denoted as CA10). Figure 2 (As shown).

[0044] Example 2

[0045] The preparation method of the biomimetic collagen corneal graft in this embodiment includes the following steps:

[0046] All experimental instruments and deionized water were sterilized in a high-temperature, high-pressure sterilizer, and then sealed with plastic wrap for later use.

[0047] A bag of collagen gel (type I collagen extracted from bovine Achilles tendon, from Guangzhou Pudao Lianxin Biotechnology Co., Ltd.) was taken out of a -20℃ freezer and thawed at room temperature for 24 hours. 5g of the collagen gel (50mg collagen content) was torn into small pieces and placed in a 500mL Erlenmeyer flask. 100mL of 0.01mol / L, pH=2 hydrochloric acid solution was added to the flask and dissolved for 16 hours to obtain a collagen solution with a concentration of 0.5mg / mL.

[0048] 20 mL of a 5% (w / w) carbodiimide crosslinking agent solution was added to the collagen solution via a separatory funnel. The crosslinking reaction was carried out at 4°C for 2 h with stirring to obtain the crosslinking product.

[0049] The cross-linking reaction product was moved to room temperature, stirred at 160 rpm for 6 h to remove the bubbles in the cross-linking reaction product; then, 2 mL of the bubble-removed cross-linking reaction product was dropped into the cavity of the female mold (the radius of curvature was 9.9 mm, and the chord length was 14 mm), the matched male mold was pressed tightly to form a closed cavity containing the cross-linking reaction product, placed at -70 °C for freezing for 70 min, taken out for melting at 6 °C for 20 h, and then freeze-dried at -15 °C for 50 h to produce a collagen material with a curvature shape; the residual hydrochloric acid in the film was washed away with deionized water, and the pH value of the solution after washing was determined in real time with a pH meter, and after reaching 7.0, drying was performed in the mold to obtain a structure biomimetic collagen corneal patch (denoted as CA5).

[0050] Example 3

[0051] The preparation method of the structure biomimetic collagen corneal patch of the present example was as follows:

[0052] All experimental instruments and deionized water were sterilized in an autoclave, and then sealed with plastic wrap for standby use.

[0053] A bag of collagen gel (type I collagen extracted from bovine Achilles tendon, from Guangzhou Pudaoliansheng Biological Technology Co., Ltd.) was taken out from a -20 °C freezer and thawed at room temperature for 24 h. 3 g of the collagen gel (the collagen content was 30 mg) was torn into small pieces and placed in a 500 mL conical flask, 100 mL of 0.01 mol / L, pH = 2 hydrochloric acid solution was added to the conical flask for dissolution for 12 h to obtain a collagen solution with a concentration of 0.3 mg / mL.

[0054] 10 mL of a cross-linking agent genipin solution with a mass content of 3% was added to the above collagen solution through a separatory funnel, and cross-linking reaction was performed at 4 °C for 2 h under stirring to obtain a cross-linking reaction product.

[0055] The cross-linking reaction product was moved to room temperature, stirred at 200 rpm for 3 h to remove the bubbles in the cross-linking reaction product; then, 2 mL of the bubble-removed cross-linking reaction product was dropped into the cavity of the female mold (the radius of curvature was 9.9 mm, and the chord length was 14 mm), the matched male mold was pressed tightly to form a closed cavity containing the cross-linking reaction product, placed at -90 °C for freezing for 50 min, taken out for melting at 2 °C for 30 h, and then freeze-dried at -25 °C for 45 h to produce a collagen material with a curvature shape; the residual hydrochloric acid in the film was washed away with deionized water, and the pH value of the solution after washing was determined in real time with a pH meter, and after reaching 7.0, drying was performed in the mold to obtain a structure biomimetic collagen corneal patch (denoted as CA3).

[0056] Test Example 1

[0057] The corneal graft prepared by cross-linking reaction without adding cross-linking agent was used as a control (denoted as CA0), and the physicochemical properties of each corneal graft were characterized in the following manner:

[0058] 1) Water content

[0059] Firstly, each corneal graft was weighed with a balance and the dry weight was recorded as W0; subsequently, each corneal graft was soaked in ultrapure water medium at room temperature, and was taken out at the set time of 5, 15, 30, 60, 120 and 240 min, respectively, and the water on the surface of each corneal graft was absorbed with filter paper before the wet weight was measured and recorded as Wt.

[0060] The water content of the corneal graft after soaking for 240 min was detected, and the results are shown in Table 1. Figure 3 As can be seen from Table 1, the water content of each structure biomimetic collagen corneal graft was 93-96%, which was similar to that of a natural cornea. Figure 3

[0061] 2) Light transmittance

[0062] Each corneal graft was soaked in ultrapure water at room temperature for 1 h and was attached to the inner wall of a cuvette. The test equipment was a UV-2600 ultraviolet visible spectrophotometer, which was adjusted to zero with a blank cuvette and was used to measure the absorbance A1 of each corneal graft in the visible light wavelength range of 400-800 nm.

[0063] The light transmittance of each corneal graft was detected, and the results are shown in Table 2. Figure 4 As can be seen from Table 2, the light transmittance of each corneal graft in the range of 800 nm visible light was high, especially the light transmittance of CA10 was as high as 90%, and the light transmittance performance was good. There was no great difference in the light transmittance and light transmittance curve in the visible light range between the tissue and the corneal graft. Figure 4

[0064] 3) Biomechanical properties

[0065] Each corneal graft was also cut into a rectangular strip of 1 cm x 0.5 cm and was soaked in ultrapure water for 1 h. Subsequently, the two ends of the wet strip were fixed in the upper and lower clamps of a DMA Q800 dynamic mechanical analyzer, and a tensile test was performed from 0 N with a loading rate of 2 N / min and a temperature of room temperature.

[0066] The biomechanical properties of each corneal graft were detected, and the results are shown in Table 3. Figure 5 As can be seen from Table 3, the biomechanical strength of each corneal graft was high, especially the biomechanical strength of CA5 and CA10 was above 1.4 MPa, and the biomechanical performance was good, which could meet the support and protection of the eyeball. Figure 5

[0067] 4) Resistance to collagenase type I degradation

[0068] ​​​First, 125 U / mg of collagenase type I was dissolved in PBS buffer (pH = 7.4) to prepare a collagenase solution with a concentration of 10 U / mL and stored in a refrigerator at 4°C. A water filter bag made of 150-mesh gauze was weighed using a balance and the mass was recorded as W0. Subsequently, each corneal graft was soaked in PBS buffer until it reached equilibrium, the surface water was absorbed with filter paper, and the total weight of each corneal graft and filter bag was measured and recorded as Wb. The filter bag containing each corneal graft was placed in a 10-mL glass tube, 5 mL of collagenase solution was added to each tube, and the tubes were placed in a shaker at 37°C and 150 rpm. The collagenase solution was replaced every 12 h. At the set times of 3, 6, 9, 12, 24, 36, 48, and 60 h, the filter bag was removed, the surface water was absorbed with filter paper, and the remaining mass was measured and recorded as Wr.

[0069] The results of the type I collagenase degradation resistance test of each corneal graft are shown in Table 1. Figure 6 Figure 6 As can be seen from Table 1, each corneal graft has good resistance to type I collagenase degradation, especially CA10, which is completely degraded until 44 h.

[0070] 5) Weight experiment

[0071] Each corneal graft was cut into a rectangular strip with dimensions of 1 cm x 0.5 cm and then soaked in ultrapure water for 1 h until it reached equilibrium. Then, one end of the wet strip was fixed to a weight, and the temperature was room temperature. Each corneal graft remained in a wet state during the test. The weight of the weight was increased until the suture was pulled out of the strip, and the maximum static force at which each corneal graft was destroyed was recorded.

[0072] The results show that CA10 can withstand a tensile force of 20.0 g (see Table 2), while CA0 can only withstand a tensile force of 0.3 g. Figure 7

[0073] 6) Surgical suture experiment

[0074] Each corneal graft was used for surgical suture, and the results showed that CA3, CA5, and CA10 have excellent suture resistance and can withstand surgical suture and tearing (see Table 3). However, CA0 is not resistant to surgical suture tearing. Figure 8

[0075] Test Example 2

[0076] ​​​The healing process of the sutured incision between the structure-bionic collagen corneal implant prepared in Example 1 and the donor cornea, the corneal epithelial repair, the growth of stromal cells, the regeneration and density of stromal nerve endings, and the stimulation and influence on the eye tissue are observed by slit lamp observation, anterior segment photography, anterior segment optical coherence tomography, and bioconfocal microscopy, etc. The eye tissue compatibility of the structure-bionic collagen corneal implant is evaluated.

[0077] The results show that the bionic collagen corneal implant can be sutured with surgical sutures, has good biocompatibility, and does not produce obvious inflammation and immune rejection reaction. Meanwhile, the corneal implant can maintain good light transmittance (see Figure 9 ).

[0078] Test Example 3

[0079] The structure-bionic collagen corneal implant prepared in Example 1 is sent to the Guangzhou Medical Device Supervision and Inspection Center (Guangdong Medical Device Quality Supervision and Inspection Institute) of the State Drug Administration for the following tests: cytotoxicity, sensitization test, intracutaneous reaction, mucous membrane irritation (eye irritation test), acute systemic toxicity, genetic toxicity, and subchronic systemic toxicity test.

[0080] The results show that the conclusions of the above tests are qualified, indicating that the structure-bionic collagen corneal implant has good safety.

[0081] Test Example 4

[0082] The structure-bionic collagen corneal implant prepared in Example 1 is used for animal tests, as follows:

[0083] First, the animals are successfully anesthetized by using pentobarbital and Xuelai injection, and then placed on the operating table. The hair around the eye is cut off, and the eye and eyelid are disinfected with povidone iodine three times. Topical anesthesia is performed with propamocaine hydrochloride eye drops, and the eyelid is opened with an eyelid speculum. A hole towel is laid on the eye surface to cover the surrounding tissue, and tobramycin eye drops are used to flush the conjunctival sac three times. The superior and inferior rectus muscles are suspended and fixed. A circular trephine is used to make a lamellar defect with a diameter of 5.0 mm and a depth of 200 μm on the rabbit eye. Then the hydrated wet corneal implant is sutured with nylon sutures to make the corneal implant completely fit the edge of the implant bed. Finally, gentamicin and dexamethasone sodium phosphate mixed injection is injected under the conjunctiva, and tobramycin dexamethasone ointment is applied on the eye surface to complete the operation.

[0084] The results show that: using the structural biomimetic collagen corneal graft for rabbit corneal lamellar transplantation, the fluorescein sodium staining of the postoperative corneal epithelialization process and the slit lamp examination of the postoperative keratoconus examination prove that the structural biomimetic collagen corneal graft can withstand surgical suture, complete the corneal cell epithelialization process in about 1 week, and the whole process is free of inflammation, neovascularization and keratoconus. In addition, the H&E staining of the corneal tissue section 6 months after the operation can be seen that the thickness has been restored, the new stromal cells grow into the wound, and the epithelium recovers to 3-4 cell layers, proving that the repair effect of the structural biomimetic collagen corneal graft is good (see Figure 10 ).

[0085] Test Example 5

[0086] The structural biomimetic collagen corneal graft prepared in Example 1 was used for clinical trials, as follows:

[0087] ① Inclusion criteria

[0088] a. Age 18-80 years old, gender unrestricted;

[0089] b. Best corrected visual acuity of the affected eye <0.4;

[0090] c. Suffering from corneal disease not involving the posterior limiting layer and endothelial layer, and planning to undergo lamellar keratoplasty;

[0091] d. The subject and / or his / her guardian can understand the purpose of the trial, show sufficient compliance with the trial program, and sign the informed consent form.

[0092] ② Exclusion criteria

[0093] a. The affected eye is combined with severe diseases affecting the ocular surface (such as severe dry eye, Sjogren's syndrome, cicatricial pemphigoid, etc.);

[0094] b. The affected eye is combined with other ocular diseases (such as glaucoma, active uveitis, scleral staphyloma, infectious endophthalmitis, retinal detachment, eyelid closure, ocular hypertension, etc.);

[0095] c. Patients with severe corneal vascularization;

[0096] d. Combined with important organ dysfunction or other severe diseases that cannot tolerate surgery, including severe coronary artery disease or cardiovascular disease without intervention, or a history of myocardial infarction within 12 months before enrollment; combined with mental illness, severe systemic disease (such as severe diabetes, with fasting blood glucose still ≥8mmol / L after taking blood sugar control measures), uncontrolled hypertension stage II and above (blood pressure is higher than 160-179 / 100-109mmHg after drug control);

[0097] e. History of allergy to commonly used ophthalmic drugs (tobramycin, dexamethasone, tacrolimus, etc.);

[0098] f. History of corneal or ocular surgery;

[0099] g. Severe allergic constitution and / or allergy to test materials;

[0100] h. Pregnant or lactating women;

[0101] i. Participation in other intervention drug clinical trials or medical device clinical trials within 3 months before enrollment;

[0102] j. Other situations where the investigator considers that the subject should not participate in this clinical trial for the benefit of the subject;

[0103] ③Termination criteria

[0104] Termination of the trial refers to the termination of the entire clinical trial before it is completed according to the protocol. The main purpose of terminating the clinical trial is to protect the rights and interests of the subjects and to ensure the quality of the clinical trial.

[0105] a. Serious safety problems occur during the clinical trial, and in order to protect the rights and interests of the subjects, the ethics committee of the clinical trial center or the principal investigator has the right to terminate the clinical trial in a timely manner;

[0106] b. The product is found to have no clinical value during the clinical trial, and the clinical trial should be terminated;

[0107] c. Significant errors are found in the clinical trial protocol during the clinical trial, making it difficult to evaluate the effects of the product; or the protocol has serious deviations in implementation, and it is difficult to evaluate the effects of the product if it continues;

[0108] d. The clinical trial institution and the investigator do not comply with the relevant laws and regulations of the clinical trial and the clinical trial protocol, and after being pointed out, they do not correct the situation, which is serious or continues.

[0109] e. The State Drug Administration orders the termination of the clinical trial for some reason.

[0110] ④ Follow-up and observation index collection time:

[0111] Screening period, 3 days ± 1 day after surgery, 7 days ± 2 days after surgery, 14 days ± 3 days after surgery, 30 days ± 7 days after surgery, 90 days ± 7 days after surgery, 180 days ± 7 days after surgery, 360 days ± 14 days after surgery.

[0112] ⑤ Dropout criteria and treatment

[0113] Drop-out definition: All subjects who signed the informed consent form and were eligible for the clinical trial, regardless of the reason for withdrawal, were considered drop-outs if they did not complete the observation period specified in the protocol.

[0114] Drop-out criteria:

[0115] a The subject was unwilling or unable to continue the clinical trial for any reason, and requested withdrawal from the clinical trial by the investigator;

[0116] b The subject developed a condition during surgery that was intolerable to the surgery, and the investigator judged that the subject should be withdrawn from the trial;

[0117] c The subject did not explicitly request withdrawal from the clinical trial, but was lost to follow-up and no longer received treatment and examination;

[0118] d The subject developed a serious adverse event (SAE), and the investigator judged that the subject should be withdrawn from the clinical trial;

[0119] e The subject had poor compliance.

[0120] Treatment of drop-outs: All drop-outs should be summarized in the clinical trial completion summary table in the case report form, and the reasons for the drop-outs should be analyzed. After the subject dropped out, the investigator should complete the evaluation items that could be completed. When the subject dropped out due to an adverse event, the investigator should take appropriate treatment measures according to the actual situation of the subject.

[0121] Postoperative follow-up observation of the in vivo healing and functional recovery of the structure of the biomimetic collagen corneal graft; the evaluation indicators include: uncorrected distance visual acuity (primary indicator), best corrected distance visual acuity (primary indicator), corneal epithelial healing degree (primary indicator), corneal curvature, graft and corneal transparency, graft and corneal thickness.

[0122] The results show that the biomimetic collagen corneal graft can withstand surgical suture, has good biocompatibility, does not produce obvious inflammation and immune rejection, and the corneal graft does not have problems such as dissolution and shedding (see Figure 11 ).

[0123] Control Example 1

[0124] Except that 10 mL of a crosslinking agent riboflavin solution with a mass content of 10% was added to the collagen solution of Example 1 through a separatory funnel, and the crosslinking reaction was carried out at a light intensity of 5 mW / cm 2 and a temperature of 4°C for 1 h, the rest was the same as Example 1.

[0125] The physicochemical properties of the corneal graft were characterized by the method of Test Example 1; the results showed that the biomechanical strength of the corneal graft prepared in this comparative example was only 0.6 MPa, and it could only withstand a pulling force of 3.8 g weight, and the biomechanical properties were poor, and could not withstand suturing and tearing of surgical thread.

[0126] Comparative Example 2

[0127] Except that the crosslinking agent glutaraldehyde solution with a mass content of 10% was used to replace the crosslinking agent citric acid solution of Example 1, the rest was the same as Example 1.

[0128] The physicochemical properties of the corneal graft were characterized by the method of Test Example 1; the results showed that the biomechanical strength of the corneal graft prepared in this comparative example was only 1.4 MPa, and it could only withstand a pulling force of 2.3 g weight, and the biomechanical properties were poor, and could not withstand suturing and tearing of surgical thread.

[0129] Comparative Example 3

[0130] The crosslinking reaction product of Example 1 was removed to room temperature, and the gas bubbles in the crosslinking reaction product were removed by stirring at a speed of 180 rpm; then, 2 mL of the crosslinking reaction product after removing the gas bubbles was dropped into the cavity of the female mold (the radius of curvature was 9.9 mm, and the chord length was 14 mm), the matching male mold was pressed tightly with the female mold to form a sealed cavity containing the crosslinking reaction product, and the collagen material with curvature shape was prepared by placing it at room temperature for 73 h; the residual hydrochloric acid in the membrane was washed away with deionized water, and the pH value of the solution after washing was determined in real time with a pH meter, and after reaching 7.0, it was dried in the mold, and the corneal graft of this comparative example was obtained (without freeze-thawing and freeze-drying).

[0131] The physicochemical properties of the corneal graft were characterized by the method of Test Example 1; the results showed that the biomechanical strength of the corneal graft prepared in this comparative example was only 0.8 MPa, and it could only withstand a pulling force of 6.5 g weight, and the biomechanical properties were poor, and could not withstand suturing and tearing of surgical thread.

[0132] Comparative Example 4

[0133] 10 mL of the crosslinking agent citric acid solution with a mass content of 10% was added to the collagen solution of Example 1 through a separatory funnel, and the crosslinking reaction was carried out at 1 ℃ for 74 h under stirring to obtain a crosslinking reaction product.

[0134] The cross-linking reaction product is removed from the room temperature, and stirred at 180 rpm for 4 h to remove the bubbles in the cross-linking reaction product; then, 2 mL of the bubble-removed cross-linking reaction product is dropped into the cavity of the female mold (the radius of curvature is 9.9 mm, and the chord length is 14 mm), the matched male mold is pressed tightly with the female mold to form a closed cavity containing the cross-linking reaction product, and a collagen material with a curved shape is prepared; the residual hydrochloric acid in the film is washed away with deionized water, the pH value of the solution after washing is determined in real time with a pH meter, and the mold is dried after the pH value reaches 7.0.

[0135] The physicochemical properties of the corneal graft are characterized by the method of Test Example 1; the results show that the biomechanical strength of the corneal graft prepared in this control example is only 1.1 MPa, and it can only withstand a tensile force of 11.4 g of weight, and the biomechanical performance is poor, and it cannot withstand suturing and tearing by surgical thread.

[0136] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacement for part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for preparing a structurally biomimetic collagen corneal graft, characterized in that, The method comprises the following steps: S1: dissolving collagen to obtain a collagen solution; S2: adding a cross-linking agent to the collagen solution to perform a cross-linking reaction; S3: performing bubble removal, molding, freeze-thaw, freeze-drying, washing and drying on the cross-linking reaction product to obtain a structure biomimetic collagen corneal implant.

2. The production method according to claim 1, characterized by, In step S1, the collagen is type I collagen extracted from bovine Achilles tendon.

3. The production method according to claim 1, characterized by, In step S1, the collagen is dissolved by using a hydrochloric acid solution; the concentration of the hydrochloric acid solution is 0.005-0.015 mol / L, and the concentration of the collagen solution is 0.1-0.5 mg / mL.

4. The method of claim 1, wherein, In step S2, the cross-linking agent is at least one selected from the group consisting of carbodiimide, genipin and citric acid.

5. The preparation method according to claim 1, characterized in that, In step S2, the cross-linking agent is added in the form of a cross-linking agent solution; the mass concentration of the cross-linking agent solution is 3-10%, and the volume ratio of the collagen solution to the cross-linking agent solution is 100 mL:10-20 mL.

6. The method of claim 1, wherein, In step S2, the temperature of the cross-linking reaction is 1-10 ℃, and the time of the cross-linking reaction is 1-3 h.

7. The preparation method according to claim 1, characterized in that, In step S3, the bubble removal comprises stirring the cross-linking reaction product at a rotating speed of 160-200 r / min for 3-6 h.

8. The method of claim 1, wherein, In step S3, the molding is performed by using a female mold and a male mold with a curvature; the curvature radius of the female mold and the male mold is 9.5-10 mm.

9. The method of claim 1, wherein, In step S3, the freeze-thawing comprises first freezing at-70 ℃ to-90 ℃ for 50-70 min, and then thawing at 2-6 ℃ for 20-30 h; the freeze-drying temperature is-15 ℃ to-25 ℃, and the freeze-drying time is 45-50 h.

10. A structured biomimetic collagen corneal onlay, comprising: The structure biomimetic collagen corneal implant is prepared by the method according to any one of claims 1-9.

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