A collagen membrane material that can be stably present in vivo for a long period of time and a method for preparing the same

Abstract

The application discloses a preparation method of a collagen membrane material which can exist stably in a body for a long time. A dense and transparent collagen membrane with a transparence of more than 95% is formed by using an electric field to control assembly of collagen fibers, the fiber structure is once physically cross-linked and solidified, and further chemical strengthening cross-linking is carried out, the cross-linking strength is improved through different reaction sites, and then the shielding of the in-vivo enzymatic cutting site is carried out. The advantage is that the collagen assembly fiber diameter can be controlled to be 30-50nm by applying high electric field energy, the collagen assembly fiber is densely and orderly arranged, the collagen assembly fiber arrangement is bionic to natural tissue collagen protein fiber arrangement, has high bionics, and can exist stably in a body for a long time.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and in particular to a collagen membrane material that can exist stably in vivo for a long time and its preparation method. Background Technology

[0002] Collagen is the most abundant protein in animal connective tissue and a major structural protein found in the human body. Collagen products can minimize acute and chronic inflammatory responses, immune rejection, and cytotoxicity, providing a good foundation for tissue repair. Simultaneously, collagen's three-dimensional network structure can mimic the natural extracellular matrix, providing an ideal scaffold for cell migration, adhesion, proliferation, and differentiation. As a substrate for implantable medical devices, such as artificial nerve sheaths, bio-isolating membranes, bone fillers, vascular stents, and heart valves, it possesses unique biological properties that can assist in the rapid repair or replacement of body tissues.

[0003] However, the rapid degradation rate of collagen membranes in vivo is a major concern in many clinical applications. The degradation rate of collagen is influenced by various factors, including the implantation site, the degree of cross-linking, and individual patient differences (such as enzyme activity). Excessive degradation can cause the scaffold to lose its structural integrity before complete tissue regeneration, leading to functional failure. For example, in thoracic, abdominal, and pelvic surgeries, organs involved include, but are not limited to, the liver, spleen, heart, lungs, and bladder. Maintaining the integrity and independence of these organs is often crucial. Implanted materials used to seal or partially replace tissue must have high tissue compatibility and be able to withstand the long-term impact of tissue fluid and enzymes. If the material degrades too rapidly, the resulting tissue defect and subsequent vacuum period can cause serious harm. Furthermore, some organs are non-regenerative, such as the human cornea. Approximately 80%–85% of the human cornea's thickness is the stroma, and the corneal stroma cannot regenerate after damage. Therefore, implanted replacement materials must stably replace corneal function long-term and must not degrade. If degradation occurs, the tissue replacement efficacy is significantly reduced. Currently, there is a lack of tissue membrane materials that can remain stable in the body for a long period of time. Summary of the Invention

[0004] This invention provides a collagen membrane material with excellent biocompatibility, high resistance to enzymatic degradation, and long-term stability in vivo through a combined process. First, a dense collagen fiber structure is obtained by controlling the assembly of collagen proteins using an electric field, while also exhibiting excellent light transmittance. Second, innovatively, by combining two cross-linking steps—ultraviolet light cross-linking and chemical cross-linking—with different reactive groups, the utilization rate of reactive groups is greatly improved, resulting in a degradation-resistant collagen membrane. Third, the enzymatic cleavage sites of collagenases are shielded; the anhydride component specifically binds to amino groups, shielding the binding domains of collagenases and further enhancing the stability of the collagen membrane.

[0005] The purpose of this invention is to provide a collagen membrane material that can exist stably in vivo for a long time and its preparation method, so as to overcome the shortcomings of the prior art.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] This application discloses a method for preparing a collagen membrane material that can exist stably in vivo for a long time, including the following steps:

[0008] S1. Substrate preparation: The collagen composite solution is assembled on a mold under the control of an electric field into a collagen membrane with dense collagen fibers and a transparency of more than 95%.

[0009] S2, One-time photocuring crosslinking: The collagen membrane obtained in step S1 under an electric field is first dehydrated with ethanol gradient to make its collagen fibers dense and solidify the fiber microstructure, and then crosslinked under a 256nm~320nm ultraviolet light source for 2~30min.

[0010] S3. Secondary chemical crosslinking: The collagen membrane after the first photocuring crosslinking in step S2 is placed in a chemical crosslinking agent solution for secondary enhanced crosslinking. After completion, the residual crosslinking agent is washed off with purified water to obtain a double-crosslinked collagen membrane.

[0011] S4. Shielding enzyme cleavage sites: The double-crosslinked collagen membrane obtained in step S3 is immersed in an acid anhydride solution with a concentration of 1~10mg / ml for 20~60 min to carry out an acylation reaction, which binds to the unblocked amino groups on the surface. After binding, it is washed with purified water to obtain the finished collagen membrane.

[0012] Preferably, in step S1, the collagen complex solution is composed of collagen, glacial acetic acid, hydrogen peroxide, polyvinylpyrrolidone K90, triethyl citrate, and water.

[0013] Preferably, step S1 includes the following sub-steps:

[0014] S11. Preparation of collagen complex solution: Dissolve collagen in a solution containing glacial acetic acid and hydrogen peroxide, then combine with film-forming agent, polyvinylpyrrolidone K90 and triethyl citrate, and stir thoroughly to obtain collagen complex solution.

[0015] S12. Electric field device assembly: Titanium sheet and platinum mesh coated sheet are used as electrode sheets, with titanium sheet as cathode and platinum mesh coated sheet as anode. They are placed in parallel and overlapping to obtain the electric field assembly device.

[0016] S13. The electric field assembly device obtained in step S12 is immersed in the collagen composite solution described above, and a high-intensity constant current with a density of 6.8~10.8 mA / cm² is applied across the electrodes.2 A dense collagen membrane composed of collagen fibers is obtained by assembling the collagen fibers in an electric field.

[0017] Preferably, in every 1000 mL of the collagen complex solution, the concentration of collagen is 0.5wt%~0.8wt%, the mass fraction of glacial acetic acid is 3.0%~4.0wt%, the mass fraction of hydrogen peroxide is 10%~15wt%, the mass fraction of polyvinylpyrrolidone K90 is 0.1wt%~1.5wt%, and the mass fraction of triethyl citrate is 0.1wt%~1.5wt%.

[0018] Preferably, in step S1, the electric field is applied for assembly for 20-30 minutes.

[0019] Preferably, in step S2, the mass fractions of ethanol for gradient dehydration are 70%, 80%, 90%, and 99% respectively, and the dehydration time for each concentration gradient is 10-20 min.

[0020] Preferably, the intensity of the ultraviolet light source in step S2 is 100~400 μw / cm. 2 The distance between the ultraviolet light source and the collagen membrane is 5~25cm.

[0021] Preferably, the chemical crosslinking agent in step S3 is at least one of genipin and EDC / NHS, and the concentration of the chemical crosslinking agent in each 1000 mL of the collagen complex solution is 0.02 wt%~0.1 wt%, crosslinking is performed at an ice bath temperature for 2~7 h.

[0022] Preferably, the anhydride mentioned in step S4 includes at least one of succinic anhydride, adipic anhydride, and maleic anhydride, and the pH of the reaction buffer system is 8.5 to 9.5.

[0023] This invention also discloses a collagen membrane material that can exist stably in vivo for a long time, and a method for preparing such a collagen membrane material.

[0024] The beneficial effects of this invention are:

[0025] 1. Compared with the traditional method of mold casting to prepare products, the present invention adopts electric field controlled collagen assembly technology. By applying a high electric field energy, the diameter of the collagen assembled fibers is controlled to be 30~50nm, and they are densely and orderly arranged, mimicking the arrangement of natural tissue collagen fibers, which has a high degree of biomimicry and makes its collagen fiber structure dense.

[0026] 2. Since some clinical applications require long-term stable in-situ service, this invention adopts an innovative double cross-linking method. Before the first cross-linking step, the collagen fiber structure is further compacted by gradient dehydration with ethanol to prevent structural disorder. Then, cross-linking is used to stabilize the structure. On the other hand, it enhances the mechanical properties and anti-swelling properties of the collagen raw materials, thereby improving the stability of the double cross-linking.

[0027] 3. Based on double cross-linking, this invention utilizes anhydride acylation of amino groups on collagen to shield biological enzyme reaction sites contained in tissue fluid, extracellular matrix, aqueous humor, and ocular surface tears, thereby further enhancing the degradation resistance of the collagen membrane. The collagen membrane can effectively serve patients for a long time, making it more convenient and advantageous in clinical use.

[0028] The features and advantages of the present invention will be described in detail through embodiments and in conjunction with the accompanying drawings. Attached Figure Description

[0029] Figure 1 In the image, (a) is an SEM image of the collagen fiber arrangement structure of Example 1, and (b) is an SEM image of the collagen fiber arrangement structure of Comparative Example 1.

[0030] Figure 2 This is a comparison diagram of the enzymatic hydrolysis stability of Examples 1, 2, and 3 and Comparative Examples 2 and 3 under collagenase.

[0031] Figure 3 In the image, a is a comparison of the in-situ state of the collagen membrane at different time points under the mouse skin, b is a comparison of the diameter and morphology of the collagen membrane at the node endpoint, and c is a comparison of HE-stained sections at different nodes.

[0032] Figure 4 In the figures, (a) is the OCT image of Comparative Example 2 implanted in the interlayer of rabbit cornea 1 day after implantation, (b) is the image of Comparative Example 2 implanted 1 month after implantation, (c) is the image of Example 2 implanted in the interlayer of rabbit cornea 1 day after implantation, and (d) is the image of Example 2 implanted 12 months after implantation.

[0033] Figure 5 This is an HE-stained section image of corneal tissue obtained from interlaminar tissue in Example 2. The morphology remains intact, with no obvious cell ingrowth. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. However, it should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of the invention. Furthermore, descriptions of well-known structures and technologies are omitted in the following description to avoid unnecessarily obscuring the concept of the invention.

[0035] This invention discloses a method for preparing a collagen membrane material that can exist stably in vivo for a long period of time, comprising the following steps:

[0036] S1. Substrate Preparation: The collagen composite solution is assembled on a mold under electric field control to form a collagen membrane substrate with dense collagen fibers and a transparency greater than 95%. The collagen composite solution is composed of collagen, glacial acetic acid, hydrogen peroxide, triethyl citrate, polyvinylpyrrolidone K90, and water. Specifically, in the collagen composite solution, the concentration of collagen is 0.5wt%~0.8wt%, the mass fraction of glacial acetic acid is 3.0%~4.0wt%, the mass fraction of hydrogen peroxide is 10%~15wt%, and the mass fractions of polyvinylpyrrolidone K90 and triethyl citrate are 0.1wt%~1.5wt%. The electric field assembly time is 20~30 min.

[0037] Specifically, step S1 includes the following sub-steps:

[0038] S11. Preparation of collagen complex solution: Dissolve collagen in a solution containing glacial acetic acid and hydrogen peroxide, then add polyvinylpyrrolidone K90 and triethyl citrate, and stir thoroughly to obtain collagen complex solution.

[0039] S12. Electric field device assembly: For example, titanium sheets and platinum mesh coated sheets are used as electrode sheets, with the titanium sheet as the cathode and the platinum mesh coated sheet as the anode, placed in parallel and overlapping to obtain an electric field assembly device.

[0040] S13. The electric field assembly device obtained in step S12 is immersed in the collagen composite solution described above, and a high-intensity constant current with a density of 6.8~10.8 mA / cm² is applied across the electrodes. 2 A dense collagen membrane composed of collagen fibers is obtained by assembling the collagen fibers in an electric field.

[0041] S2, One-time photocuring crosslinking: The collagen membrane substrate obtained in step S1 under an electric field is first dehydrated with ethanol gradient to make its collagen fibers dense and solidify the fiber microstructure, and then placed under a 256nm~320nm ultraviolet light source for crosslinking for 2min~30min to further stabilize the structure.

[0042] S3. Secondary chemical crosslinking: After primary crosslinking, the collagen membrane is demolded and placed in a chemical crosslinking agent solution of a certain concentration for secondary enhanced crosslinking; after completion, the residual crosslinking agent is washed off to obtain the double-crosslinked collagen membrane.

[0043] S4. Shielding enzyme cleavage reaction sites: The double-crosslinked collagen membrane is immersed in an acid anhydride solution with a concentration of 1 mg / ml~10 mg / ml for 20~60 min to undergo acylation reaction, which binds to the unblocked amino groups on the surface. After the reaction, it is washed.

[0044] In one feasible embodiment, the ethanol gradient dehydration mass fractions in step S2 are 70%, 80%, 90%, and 99% respectively, with a dehydration time of 10-20 min for each concentration gradient; the intensity of the ultraviolet light source is 100-400 μw / cm². 2 The distance between the ultraviolet light source and the collagen membrane is 5~25cm.

[0045] In one feasible embodiment, the chemical crosslinking agent in step S3 is genipin, EDC / NHS, or one of them, with a solution concentration of 0.02 wt% to 0.1 wt%.

[0046] In one feasible embodiment, the chemical crosslinking environment in step S3 is crosslinking under an ice bath, and the crosslinking time is 2~7h.

[0047] In one feasible embodiment, the anhydride mentioned in step S4 is one or more of succinic anhydride, adipic anhydride, and maleic anhydride, and the pH of the reaction buffer system is 8.5~9.5.

[0048] Example 1:

[0049] S1. Preparation of collagen membrane substrate:

[0050] S11: Collagen is dissolved in a 3.0 wt% glacial acetic acid solution, hydrogen peroxide is added at a mass fraction of 10 wt%, 0.1 wt% polyvinylpyrrolidone K90 and 0.5 wt% triethyl citrate are added, and the collagen concentration is 0.5 wt%, yielding 1000 mL of solution. After thorough mixing, pre-cool for later use.

[0051] S12: A titanium sheet and a platinum mesh coated sheet electrode with dimensions of 20×20cm are placed parallel to each other in the electric field assembly tank as the cathode and anode, respectively.

[0052] S13: Pour the collagen solution along the edge into the assembly tank, then apply current to the electrodes at a current density of 6.8 mA / cm². 2 The electric field assembly time is 30 minutes.

[0053] S2, One-time photocuring crosslinking: The collagen membrane obtained in step S1 under an electric field is first dehydrated using a gradient of 70%, 80%, 90%, and 99% ethanol for 10 minutes each, to densify the collagen fibers and solidify the fiber microstructure. Then, it is placed under a 256nm ultraviolet light source, 5cm away from the membrane, at 100μw / cm. 2 Crosslinking at the specified intensity for 2 minutes further stabilizes the structure.

[0054] S3. Secondary chemical crosslinking: After primary crosslinking, the collagen membrane is demolded and placed in a 0.02 wt% genipin solution for secondary enhanced crosslinking for 7 hours under ice bath conditions. After completion, the residual crosslinking agent is washed off with purified water to obtain the double-crosslinked collagen membrane.

[0055] S4. Shielding enzyme cleavage reaction sites: The double-crosslinked collagen membrane is soaked in succinic anhydride and maleic anhydride at a total concentration of 1 mg / ml under the condition of pH 8.5 for 20 min to carry out acylation reaction, which binds to the unblocked amino groups on the surface. After the reaction, it is washed with purified water.

[0056] After completion, the samples were sealed in aluminum foil and sterilized with electron beam to obtain the finished collagen membrane.

[0057] Example 2:

[0058] S1. Preparation of collagen membrane substrate:

[0059] S11: Collagen was dissolved in a 4.0 wt% glacial acetic acid solution with 15 wt% hydrogen peroxide. 0.2 wt% polyvinylpyrrolidone K90 and 1.5 wt% triethyl citrate were added, resulting in a collagen concentration of 0.8 wt%, yielding 1000 mL of solution. After thorough mixing, the solution was pre-cooled for later use.

[0060] S12: A titanium sheet and a platinum mesh coated sheet electrode with dimensions of 15×15cm are placed parallel to each other in the electric field assembly tank as the cathode and anode, respectively.

[0061] S13: Pour the above collagen solution along the edge into the assembly tank, then apply current to the electrodes at a current density of 8.0 mA / cm². 2 The electric field assembly time is 20 minutes.

[0062] S2, One-time photocuring crosslinking: The collagen membrane obtained in step S1 under an electric field is first dehydrated with a gradient of 70%, 80%, 90%, and 99% ethanol for 20 minutes each, to densify the collagen fibers and solidify the fiber microstructure. Then, it is placed under a 320nm ultraviolet light source, with the membrane 25cm away from the light source, at 400μw / cm. 2 Crosslinking at high intensity for 10 minutes further stabilizes the structure;

[0063] S3. Secondary chemical crosslinking: After primary crosslinking, the collagen membrane is demolded and placed in a 0.1 wt% EDC / NHS solution for secondary enhanced crosslinking in an ice bath for 2 hours. After completion, the residual crosslinking agent is washed off to obtain the double-crosslinked collagen membrane.

[0064] S4. Shielding enzyme cleavage reaction sites: The double-crosslinked collagen membrane was immersed in a reaction buffer system of succinic anhydride, maleic anhydride and adipic anhydride with a total concentration of 8 mg / ml at pH 9.5 for 40 min to carry out acylation reaction, binding with the unblocked amino groups on the surface. After the reaction, the membrane was washed with purified water.

[0065] After completion, the samples were sealed in aluminum foil and sterilized with electron beam to obtain the finished collagen membrane.

[0066] Example 3:

[0067] S1. Preparation of collagen membrane substrate:

[0068] S11: Collagen was dissolved in a 3.0 wt% glacial acetic acid solution with 12 wt% hydrogen peroxide. 1.5 wt% polyvinylpyrrolidone K90 and 0.1 wt% triethyl citrate were added, resulting in a collagen concentration of 0.6 wt%, yielding 1000 mL of solution. After thorough mixing, the solution was pre-cooled for later use.

[0069] S12: A titanium sheet and a platinum mesh coated sheet electrode with dimensions of 20×20cm are placed parallel to each other in the electric field assembly tank as the cathode and anode, respectively.

[0070] S13: Pour the collagen solution along the edge into the assembly tank, then apply current to the electrodes at a current density of 10.8 mA / cm². 2 The electric field assembly time is 20 minutes.

[0071] S2, One-time photocuring crosslinking: The collagen membrane obtained in step S1 under an electric field is first dehydrated using a gradient of 70%, 80%, 90%, and 99% ethanol for 15 minutes each, to densify the collagen fibers and solidify the fiber microstructure. Then, it is placed under a 256nm ultraviolet light source, with the membrane 15cm away from the light source, at 200μw / cm. 2 Crosslinking at high intensity for 30 minutes further stabilizes the structure;

[0072] S3, Secondary chemical crosslinking: After primary crosslinking, the collagen membrane is demolded and placed in a 0.05 wt% genipin solution for secondary enhanced crosslinking for 3 hours in an ice bath; after completion, the residual crosslinking agent is washed off to obtain the double-crosslinked collagen membrane.

[0073] S4. Shielding enzyme cleavage reaction sites: The double-crosslinked collagen membrane was soaked in succinic anhydride with a total concentration of 10 mg / ml under the condition of pH 9.0 for 60 min to carry out the acylation reaction, which binds to the unblocked amino groups on the surface. After the reaction, it was washed with purified water.

[0074] After completion, the samples were sealed in aluminum foil and sterilized with electron beam to obtain the finished collagen membrane.

[0075] Comparative Example 1:

[0076] Comparative Example 1 is based on Example 1, but without undergoing gradient dehydration with ethanol before the first UV crosslinking, and compares the degree of tightness of collagen fiber arrangement obtained with that of Example 1.

[0077] Comparative Example 2:

[0078] Comparative Example 2, based on Example 2, did not involve a second chemical cross-linking but only a single UV cross-linking. The in vitro enzymatic resistance of the resulting collagen membrane was compared with that of the Example 2.

[0079] Comparative Example 3:

[0080] Comparative Example 3 is based on Example 1, but without masking the enzyme cleavage reaction sites, that is, without using succinic anhydride and maleic anhydride to react the amino groups on the collagen surface.

[0081] Based on the above embodiments and comparative examples, the specific tests are as follows:

[0082] Experiment 1: SEM testing of collagen fiber structure

[0083] Five samples were selected from each of Example 1 and Comparative Example 1. The samples were dehydrated using a gradient of ethanol at 70%, 90%, and 100% for 5 minutes each time. The ethanol was then replaced with isoamyl acetate, and the samples were further dried using a CO2 critical point dryer. The collagen membrane samples were treated twice using a Hitachi S-3800 15kV sprayer for observation. Representative samples were selected for analysis. (See Appendix) Figure 1 After being dehydrated by a gradient of ethanol in front of ultraviolet light, the collagen fiber structure becomes significantly denser. However, without prior ethanol dehydration, the collagen fibers have large gaps, a loose structure, and poor biomimetic properties.

[0084] Experiment 2: Mechanical property testing of collagen membranes:

[0085] Mechanical properties were tested using collagen matrix implants from Examples 1-3 and Comparative Examples 1 and 2. Samples were cut into 6×30mm pieces, and the tensile testing machine was set to a tensile speed of 100mm / min. Each test sample had n=8 samples. As shown in Table 1, the materials exhibited strong tensile strength. In Comparative Example 1, because no ethanol dehydration reaction was performed before the first crosslinking, the collagen fibers were not densely packed, resulting in poor mechanical properties. In Comparative Example 2, because only single chemical crosslinking was performed, the tensile strength generally decreased by nearly 50%.

[0086] Table 1

[0087]

[0088] Experiment 3: Light Transmittance Test

[0089] The collagen membranes from Examples 1-3 and Comparative Example 1 were used to conduct light transmittance tests. The transmittance of the collagen implants at 600 nm was measured using a transmittance meter, and the diameter of the collagen fibers was scanned using an electron microscope. The results are shown in Table 2. The collagen fibers were not dehydrated using an ethanol gradient before crosslinking, resulting in larger gaps between the collagen fibers. This led to significant scattering and reduced light transmittance, limiting their application in scenarios where tissue light transmittance is important.

[0090] Table 2 Transmittance Test Data

[0091]

[0092] Experiment 4: In vitro enzymatic hydrolysis test of collagen membrane

[0093] The in vitro degradation rate of the collagen membrane products from Examples 1-3 and Comparative Examples 2-3 was evaluated using type I collagenase (derived from Clostridium histolyticum). The collagen membrane thickness was 300 μm, and the dimensions were 1 × 1 cm. The hydrolysate was replaced every 8 hours to maintain enzyme activity. The entire hydrolysis process was conducted in a 37°C constant temperature water bath. The collagen membranes from Examples 1-3, which were doubly cross-linked and had their cleavage sites shielded with anhydride, showed significantly higher resistance to type I collagenase degradation than those from Comparative Examples 2-3, taking nearly 200 hours to completely degrade. (See Appendix). Figure 2 Comparative Example 2, which did not undergo secondary chemical cross-linking, had poor resistance to enzymatic hydrolysis and degraded in about 24 hours. Comparative Example 3, which did not have its enzyme cleavage sites shielded, was completely degraded in less than 30 hours. Therefore, both double cross-linking and shielding of enzyme cleavage sites can significantly improve the product's resistance to enzymatic hydrolysis.

[0094] Experiment 5: Subcutaneous implantation experiment in mice:

[0095] Before the experiment, 20 mice were anesthetized with sodium pentobarbital injection. The fur on the backs of the mice was shaved, and a 2cm incision was made in the skin. Subcutaneous tissue was dissected using a blunt instrument. Collagen membrane samples were photographed for diameter and morphology before implantation. One sample was implanted into each mouse. Mice were housed individually. Postoperatively, the animals' physiological status and adverse events were observed daily. Physiological status included physical appearance, behavior, body temperature, local irritation, fecal characteristics, and the presence of redness, swelling, or infection at the surgical site. One week after surgery, appropriate antibiotics were added to the diet to prevent infection. Samples were collected at each endpoint (1, 3, 6, and 12 months). After removal, the collagen membranes were rinsed briefly with saline and photographed for morphology.

[0096] Materials obtained for each experimental node are listed in the appendix. Figure 3Figure a shows the collagen membrane in situ under the skin; figure b shows photographs of its diameter and morphology after removal; and figure c shows HE-stained sections at different nodes. No obvious macrophages, granulocytes, lymphocytes, or fibrous encapsulation were observed, indicating excellent biocompatibility after product implantation. Furthermore, even after subcutaneous implantation in mice for up to one year, the collagen membrane morphology showed no significant change, demonstrating extremely high stability and its ability to function as a tissue barrier membrane.

[0097] Experiment 6: Collagen membrane implantation test in rabbit corneal stroma

[0098] Eight experimental rabbits underwent SMILE surgery in their left eyes after being anesthetized with dexmedetomidine hydrochloride (Domexin) and sedative according to their weight. The left eye received a collagen membrane graft from Example 2, while the left eye received a collagen membrane graft from Comparative Example 2 as a control. The corneal cap thickness was 150 μm, the corneal cap diameter was 7.5 mm, the incision angle was 90°, and the incision length was 2.5 mm. After laser scanning, the negative pressure suction was removed, the corneal incision was dissected, and the anterior and posterior surfaces of the native stromal tissue were sequentially separated and removed. A 7 mm diameter, 100 μm thick collagen membrane was inserted into the capsular bag using forceps and centered, without sutures. Parameters were kept consistent between the left and right eyes. All rabbits wore corneal bandage lenses post-surgery and received levofloxacin and dexamethasone-tobramycin eye drops 3-4 times daily for post-operative care. Medication was discontinued after 4 weeks if no abnormalities were observed. OCT scans of the animals at different post-operative time points are shown in the appendix. Figure 4 (a) is an OCT image of Comparative Example 2 implanted in the interlaminar space of rabbit cornea one day after implantation. (b) is an image of Comparative Example 2 implanted one month after implantation, showing that the collagen membrane has been completely degraded, indicating that no secondary chemical cross-linking has been performed. The product has poor resistance to enzymatic degradation and cannot maintain its intended efficacy in the corneal tissue. (c) is an OCT image of Example 2 implanted in the interlaminar space of rabbit cornea one day after implantation. (d) is an image of Example 2 implanted 12 months after implantation, showing good morphology, no obvious scarring, and no obvious signs of cell ingrowth. At the 12-month mark, the cornea was removed for tissue staining and sectioning, as shown in the attached image. Figure 5 As shown, the collagen membrane remains intact and still exhibits a dense state.

[0099] 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 within the protection scope of the present invention.

Claims

1. A method for preparing a collagen membrane material that can exist stably in vivo for a long period of time, characterized in that: Includes the following steps: S1. Substrate preparation: The collagen composite solution is assembled on a mold under the control of an electric field into a collagen membrane with dense collagen fibers and a transparency of more than 95%. S2, One-time photocuring crosslinking: The collagen membrane obtained in step S1 under an electric field is first dehydrated with an ethanol gradient, wherein the mass fraction of the ethanol gradient dehydration is 70%, 80%, 90%, and 99% respectively, and the dehydration time for each concentration gradient is 10~20 min, so that the collagen fibers are dense and the fiber microstructure is cured, and then crosslinked under a 256nm~320nm ultraviolet light source for 2~30 min; S3. Secondary chemical crosslinking: The collagen membrane after the first photocuring crosslinking in step S2 is placed in a chemical crosslinking agent solution for secondary enhanced crosslinking. After completion, the residual crosslinking agent is washed off with purified water to obtain a double-crosslinked collagen membrane. S4. Shielding enzyme cleavage sites: The double-crosslinked collagen membrane obtained in step S3 is immersed in an acid anhydride solution with a concentration of 1~10mg / ml for 20~60 min to carry out an acylation reaction, which binds to the unblocked amino groups on the surface. After binding, it is washed with purified water to obtain the finished collagen membrane.

2. The method for preparing a collagen membrane material that can exist stably in vivo for a long time according to claim 1, characterized in that: In step S1, the collagen complex solution is composed of collagen, glacial acetic acid, hydrogen peroxide, polyvinylpyrrolidone K90, triethyl citrate and water.

3. The method for preparing a collagen membrane material that can exist stably in vivo for a long time according to claim 2, characterized in that: Step S1 includes the following sub-steps: S11. Preparation of collagen complex solution: Dissolve collagen in a solution containing glacial acetic acid and hydrogen peroxide, then combine with film-forming agent, polyvinylpyrrolidone K90 and triethyl citrate, and stir thoroughly to obtain collagen complex solution. S12. Electric field device assembly: Titanium sheet and platinum mesh coated sheet are used as electrode sheets, with titanium sheet as cathode and platinum mesh coated sheet as anode. They are placed in parallel and overlapping to obtain the electric field assembly device. S13. The electric field assembly device obtained in step S12 is immersed in the collagen composite solution described above, and a high-intensity constant current with a density of 6.8~10.8 mA / cm² is applied across the electrodes. 2 A dense collagen membrane composed of collagen fibers is obtained by assembling the collagen fibers in an electric field.

4. The method for preparing a collagen membrane material that can exist stably in vivo for a long time according to claim 3, characterized in that: In every 1000 mL of the collagen complex solution, the concentration of collagen is 0.5wt%~0.8wt%, the mass fraction of glacial acetic acid is 3.0%~4.0wt%, the mass fraction of hydrogen peroxide is 10%~15wt%, the mass fraction of polyvinylpyrrolidone K90 is 0.1wt%~1.5wt%, and the mass fraction of triethyl citrate is 0.1wt%~1.5wt%.

5. The method for preparing a collagen membrane material that can exist stably in vivo for a long time according to claim 1, characterized in that: In step S1, the electric field is applied for assembly for 20-30 minutes.

6. The method for preparing a collagen membrane material that can exist stably in vivo for a long time according to claim 1, characterized in that: In step S2, the intensity of the ultraviolet light source is 100~400 μw / cm. 2 The distance between the ultraviolet light source and the collagen membrane is 5~25cm.

7. The method for preparing a collagen membrane material that can exist stably in vivo for a long time according to claim 1, characterized in that: In step S3, the chemical cross-linking agent is at least one of genipin or EDC / NHS, and the concentration of the chemical cross-linking agent in each 1000 mL of the collagen complex solution is 0.02 wt% to 0.1 wt%. Cross-linking is performed at an ice bath temperature for 2 to 7 h.

8. The method for preparing a collagen membrane material that can exist stably in vivo for a long time according to claim 1, characterized in that: The anhydride mentioned in step S4 includes at least one of succinic anhydride, adipic anhydride, and maleic anhydride, and the pH of the reaction buffer system is 8.5~9.

5.

9. A collagen membrane material that can exist stably in vivo for a long period of time, characterized in that: The preparation method of a collagen membrane material that can exist stably in vivo for a long time, as described in any one of claims 1-8, is adopted.

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