A collagen-based hydrogel for corneal injury repair and its uses

Abstract

The present invention provides a collagen-based hydrogel for corneal injury repair. First, type I collagen is prepared into a thermodynamically self-assembled collagen hydrogel Col, which is then immersed in a polar molecule solution to prepare a collagen-based hydrogel Col-Gly. The present invention also provides a collagen-based hydrogel for corneal injury repair, which is prepared from a collagen-based hydrogel Col-Gly, a polar molecule solution, a crosslinking agent, and water to form a collagen-based hydrogel Col-Gly-OX. The collagen-based hydrogel of the present invention is used for corneal injury repair and has high light transmittance and excellent mechanical strength.

Description

Technical Field

[0001] The present invention relates to a collagen-based artificial cornea substitute, provides a collagen-based hydrogel for corneal injury repair and its use, and belongs to the field of biomedical materials. Background Art

[0002] The cornea is the transparent tissue at the front of the eye and plays an important role in regulating refractive function and preventing foreign objects from invading. Corneal-related diseases may cause vision loss in patients, and in severe cases, it may lead to vision loss and the risk of blindness. Corneal transplantation is considered the gold standard for restoring vision in patients with corneal blindness. However, only 1 in 70 patients globally can receive a corneal transplantation surgery. Therefore, developing a corneal substitute with simple manufacturing and good biocompatibility is very important for alleviating the shortage of corneal donors.

[0003] Currently, a variety of biomaterials have been developed as bionic scaffolds for corneal injury repair, including natural macromolecules (such as collagen, gelatin, hyaluronic acid, chitosan, alginate, etc.), synthetic polymers (such as polyethylene glycol (PEG), 2-hydroxyethyl methacrylate (PHEMA), etc.) or their composite materials. Although these materials have properties similar to those of natural corneas, only a few of them can be applied clinically due to reasons such as complex manufacturing processes, low transparency, insufficient mechanical stability after application, and poor biological binding to tissues.

[0004] Application No.: CN202210786167.X, Invention Title: Preparation Method and Application of a Thermosensitive Corneal Repair Hydrogel with Biological Activity, discloses a preparation method of a thermosensitive corneal repair hydrogel with biological activity, and the steps are as follows: S1, collecting biological tissues; S2, virus inactivation; S3, repeated freezing and thawing;

[0005] S4. Tissue decellularization; S5. Preparation of gel: First, place the tissue obtained in step S4 at -100°C to -40°C for pre-freezing for 12 to 18 hours, and then freeze-dry it. Then, grind the freeze-dried tissue into acellular particles smaller than 1 mm, perform enzymatic hydrolysis, and after completion, adjust the pH to 6.0 to 7.5 with an alkaline solution, neutralize the isotonic solution, and adjust the concentration; S6. Add an anti-corrosion system, and that's it. The present invention adopts the above-mentioned preparation method and application of a thermosensitive corneal repair hydrogel with biological activity. The prepared hydrogel contains rich collagen, elastin, and mucopolysaccharides, effectively inhibits the corneal lesion process caused by physical or chemical damage, and self-crosslinks to form a "biofilm" under body temperature conditions to play the role of an object barrier, thereby promoting the regeneration and repair of damaged corneas. Application number: CN201610557778.1, Invention title: Preparation method of a corneal repair implant with biological activity and a corneal repair implant. It discloses a preparation method of a corneal repair implant with biological activity and a corneal repair implant, which solves the technical problem of the lack of corneal donors. It includes: preparing a collagen swelling solution; based on the collagen swelling solution, preparing a collagen membrane; under sterile conditions, adding a surface-functionalized modified substance to the surface of the collagen membrane in a 3D printing or coating manner to form a corneal repair implant with biological activity. The surface-functionalized modified substance is made of fine particles prepared from the corneal stroma containing active cells with the diseased part removed from the patient's own body, which can improve the biological and biological stability of the high-strength collagen membrane, improve the physiological function of the cornea, make the modified collagen membrane have good transparency and water content after being implanted into the patient's eye, can recover transparency faster clinically, has good biocompatibility, and provides a good scaffold for the corneal cell culture of corneal transplant recipients.

[0006] Collagen is the main structural component of the native corneal stroma and is therefore the first choice for making corneal substitutes. In vivo, collagen triple-helix molecules can self-assemble orderly according to endogenous cues (such as proteoglycans, type V collagen), ranging from nanometers to micrometers. In vitro, collagen fibers can also self-assemble and reorganize through hydrogen bonds, electrostatic, and hydrophilic-hydrophobic interactions at 37°C and neutral pH, but this thermodynamically driven process often cannot be controlled and exhibits poor transparency and mechanical properties. The main reason for these results is the excessive aggregation of collagen fibers during the self-assembly process. Although chemical crosslinking can improve the light transmittance and mechanical properties of the collagen scaffold to a certain extent, the limited mechanical integrity caused by crosslinking may lead to poor performance of the transplant suture.

[0007] Recently, researchers have carried out studies on regulating the self-assembly behavior of collagen through various external cues (physical and chemical signals), aiming to mimic the self-assembly behavior of collagen in vivo. For example, Majumdar et al. screened cyclodextrins of different sizes and chemical functions to regulate the self-assembly of collagen in order to create a biomimetic that mimics the natural corneal structure. Although this hydrogel has a certain degree of transparency and sutureability, the complex dehydration process and limited performance limit its application as a corneal substitute. Lei et al. reported a method for electrochemically dynamically assembling type I collagen into a highly transparent structural material. Although this method shows good application prospects in corneal tissue regeneration, it requires strict conditions (requiring an external electric field and a customized mold), and in addition, a cross-linking agent, glutaraldehyde, needs to be used, and glutaraldehyde also has a certain degree of cytotoxicity. The basis of these methods is to actively regulate the self-assembly behavior of collagen to manufacture transparent collagen-based materials by restricting the excessive aggregation of collagen.

[0008] In the leather-making field, there is a solution for dissolution and regeneration. The self-assembled collagen will break the self-assembly process under the action of external factors (such as ionic liquids and polar molecules), resulting in a change in fiber diameter, and this change in fiber diameter provides the possibility for designing transparent collagen hydrogels. In addition, the use of new cross-linking agents is also expected to solve the problem that traditional cross-linking agents are brittle and difficult to be used for corneal suturing.

[0009] In summary, as one of the main components of the cornea, collagen is an ideal building block for corneal defect repair. How to design a collagen-based artificial corneal substitute with high light transmittance and excellent mechanics is of great significance for alleviating the shortage of corneal donors. Summary of the Invention

[0010] The purpose of the present invention is to provide a collagen-based hydrogel with high light transmittance and excellent mechanical strength for corneal injury repair and its preparation method. The collagen-based hydrogel has a light transmittance of up to 95%, and after chemical cross-linking, the gel can have excellent toughness while maintaining high light transmittance, meeting the requirements of corneal transplantation suturing, and thus has important significance in the field of corneal injury repair.

[0011] The present invention provides a collagen-based hydrogel for corneal injury repair, which first prepares type I collagen into a thermodynamically self-assembled collagen hydrogel Col and immerses it in a polar molecule solution to prepare a collagen-based hydrogel Col-Gly; the volume ratio of the polar molecule to water is: 0 - 100%.

[0012] Among them, the type I collagen is derived from fetal bovine skin or other livestock and poultry source animal tissues;

[0013] The volume ratio of the polar molecular solution to the collagen hydrogel Col is greater than 10;

[0014] The polar molecules are glycerol, urea, Ca 2+ , ionic liquids, and the volume ratio of the polar molecule to water is: 0:5, 1:4, 2:3, 3:2, 4:1, 5:0;

[0015] The soaking time is 1 - 60 min.

[0016] Further preferably, the volume ratio of glycerol to water is: 4:1; the soaking time is 30 min.

[0017] The collagen-based hydrogel Col-Gly is a highly transparent collagen-based hydrogel.

[0018] Among them, the preparation method of the thermodynamically self-assembled collagen hydrogel Col is:

[0019] a. Dissolve type I collagen derived from fetal bovine skin in a 0.1 - 0.5 M glacial acetic acid solution, and the concentration of type I collagen is 5 - 15 mg / ml;

[0020] b. After complete dissolution of the type I collagen solution, adjust the pH to 7.1 - 8 at 2 - 4 °C using a 1 - 5 M sodium hydroxide solution; then remove internal bubbles by centrifugation at 3000 - 9000 r / min with a high-speed centrifuge at 2 - 4 °C;

[0021] c. After taking it out, transfer the collagen solution to a ring made of polytetrafluoroethylene using a syringe, and place it in a constant temperature incubator at 30 °C - 45 °C, and incubate for 20 - 60 min to obtain the thermodynamically self-assembled collagen hydrogel Col; preferably, the temperature in the constant temperature incubator is 37 °C.

[0022] In the present invention, the thermodynamically self-assembled collagen gel is treated with polar molecules. By disrupting the interaction between collagen fiber molecules, the structure of the fibers is changed, thereby obtaining excellent optical properties determined by the structure, including high transparency and low haze properties. Since the mechanical strength of the collagen gel after treatment with polar molecules still cannot meet the requirements of corneal suture, and the high light transmittance obtained by simply treating with polar molecules cannot be maintained in water for a long time, further cross-linking is required.

[0023] The present invention also provides a collagen-based hydrogel for corneal injury repair, which is prepared from the collagen-based hydrogel Col-Gly, a polar molecular solution, a crosslinking agent, and water into a collagen-based hydrogel Col-Gly-OX; wherein the volume of the polar molecular solution is 30%-60%, and the concentration of the crosslinking agent is 2%-20%; the polar molecule is glycerol, and the crosslinking agent is oxazolidine; the concentration range of the collagen-based hydrogel Col-Gly is 5-15 mg / ml.

[0024] Among them, the volume of the polar molecular solution is 80%, and the concentration of the crosslinking agent is 10%.

[0025] Among them, the preparation method is soaking, and the soaking time is 2-30 min.

[0026] Among them, the hydrogel contains compounds:

[0027] The hydrogel contains compounds:

[0028]

[0029] The present invention provides the use of the collagen-based hydrogel in the preparation of a material for corneal injury repair.

[0030] In the present invention, a novel crosslinking agent is used to chemically crosslink the fibers in the transparent collagen hydrogel, and a collagen-based hydrogel (Col-Gly-GA) with both high light transmittance and excellent mechanical properties is obtained. The treatment with the polar molecule glycerol significantly reduces the size of the collagen fibers, and the reduction in fiber size makes the collagen gel have a light transmittance as high as 95%, which is much higher than the light transmittance requirement of artificial corneal substitutes. The use of the crosslinking agent oxazolidine enables the collagen-based hydrogel to have good toughness while maintaining high mechanical strength, meeting the requirements of corneal suturing. Therefore, it is a good artificial corneal substitute suitable for corneal transplantation.

[0031] Compared with the prior art, the collagen-based hydrogel for corneal injury repair provided by the present invention, which has high light transmittance and excellent mechanical strength, and its preparation method have the following beneficial technical effects:

[0032] (1) The collagen-based hydrogel with high light transmittance and excellent mechanical strength for corneal injury repair provided by the present invention. After the traditional collagen hydrogel is self-assembled, due to the excessive and disordered aggregation of collagen fibers, the mechanical properties and light transmittance of the collagen gel are very poor, unable to meet the requirements of corneal transplantation. Soaking in glycerol solution can endow the collagen hydrogel with a light transmittance as high as 95%. By testing the structure of collagen, it is found that after being treated with glycerol, the diameter of collagen fibers of about 100 nanometers is reduced to 10 nanometers. Specifically, glycerol can partially disrupt the intermolecular interactions (such as hydrogen bonds and hydrophobic interactions) between collagen fibers, thereby decomposing the over-focused collagen fibers into nanoscale fibers. Generally, the whole process only takes a few minutes, and the size of collagen can reach the nanoscale. The cornea, as an important part of the eyeball, plays an important role in refractive adjustment. Therefore, designing materials with high light transmittance is a prerequisite for designing artificial cornea substitutes. The method for improving light transmittance provided by the present invention is the highest among collagen-based hydrogels and is of great significance in the field of corneal injury repair.

[0033] (2) The collagen-based hydrogel with high light transmittance and excellent mechanical strength for corneal injury repair provided by the present invention. Another important problem faced by collagen-based hydrogels during application is low strength, unable to meet the requirements of corneal suturing. The use of traditional cross-linking agents, such as glutaraldehyde, although can greatly improve the mechanical strength of collagen gels, but will lead to an increase in their brittleness and easy fracture, unable to meet the requirements of corneal suturing. In this study, a new type of cross-linking agent - oxazolidine is used. It is a cyclic cross-linking agent that can interact with amino groups between collagen fibers in covalent and non-covalent ways. Therefore, while improving the mechanical strength, the collagen hydrogel can maintain high toughness. It is found through testing that its elongation at break can reach 60%, and the suture force can reach 0.5 N, meeting the requirements of corneal suturing. Therefore, it has broad prospects in the application of artificial corneal transplantation.

[0034] (3) The collagen-based hydrogel with high light transmittance and excellent mechanical strength for corneal injury repair provided by the present invention. Treating the self-assembled collagen gel with glycerol will disrupt the hydrogen bond interaction between collagen fiber molecules, reducing the mechanical strength of the collagen gel, and it can adapt to surfaces of various shapes. This shape plasticity can establish collagen-based membrane materials of various shapes. It should be noted that the cornea is a transparent tissue with a certain curvature. Therefore, the shape-plastic collagen-based hydrogel provided by the present invention has important application value in the design of curvature-type artificial cornea substitutes.

[0035] (4) The collagen-based hydrogel with high light transmittance and excellent mechanical strength provided by the present invention for corneal injury repair. The collagen-based hydrogel with curvature proposed by the present invention can maintain the curvature after freeze-drying. Interestingly, when immersed in water again, the gel film can quickly absorb water and swell, restoring to the state before freeze-drying, and the surface morphology is smooth. In the face of special conditions such as war, this kind of artificial cornea substitute that can be quickly used is of great significance for corneal transplantation surgery under special conditions. Description of the Drawings

[0036] Figure 1 Concentration screening test of the polar molecule glycerol solution of the present invention

[0037] Figure 2 It is the change in the light transmittance of collagen after glycerol treatment in the self-assembly process in Example 1. Among them, Figure A is a photo of the light transmittance during the process of glycerol treatment of self-assembled collagen gel, and Figure B is the curve of the light transmittance of the collagen gel after glycerol treatment in the self-assembly process changing with time.

[0038] Figure 3 It is the microscopic morphology diagrams of the thermodynamically self-assembled collagen hydrogel (Col) and the collagen hydrogel after glycerol treatment (Col-Gly) in Example 1. Among them, Figure A is the microscopic morphology of the thermodynamically self-assembled collagen gel, mainly including TEM, AFM and CLSM data. Among them, Figure B is the microscopic morphology of the collagen after glycerol treatment in the self-assembly process, mainly including TEM, AFM and CLSM data.

[0039] Figure 4 It is the mechanism exploration diagram of glycerol treatment improving the light transmittance of the collagen hydrogel in Example 1. Among them, Figure A is the change in the crystalline region observed by polarized light microscopy after glycerol treatment of the collagen gel, Figure B is the two-dimensional small-angle scattering diagram of the collagen gel before and after glycerol treatment, Figure C is the statistical chart of the two-dimensional small-angle scattering of the collagen gel before and after glycerol treatment, and Figure D is the fiber-interstitial size diagram calculated by X-ray diffraction.

[0040] Figure 5 It is the structural analysis diagram of the collagen gel before and after glycerol treatment in Example 1. Figure A is the circular dichroism spectrum diagram of the collagen gel before and after glycerol treatment, and Figure A is the Fourier transform infrared spectrum diagram of the collagen gel before and after glycerol treatment.

[0041] Figure 6 It is the schematic diagram of the collagen gel after chemical cross-linking and glycerol treatment in Example 1. Figure A is the chemical reaction formula of glutaraldehyde cross-linking Col-Gly. Figure B is the chemical reaction formula of oxazolidine cross-linking Col-Gly.

[0042] Figure 7For the optical properties of the collagen gel after chemical crosslinking with glycerol in Example 3, Figure A shows the change in transmittance of the collagen gel after chemical crosslinking with glycerol, and Figure B shows the change in haze value of the collagen gel after chemical crosslinking with glycerol.

[0043] Figure 8 For the mechanical properties of the collagen gel after chemical crosslinking with glycerol in Example 3, Figure A shows the stress-strain curve of the collagen gel after chemical crosslinking with glycerol, and Figure B shows the suture strength curve of the collagen gel after chemical crosslinking with glycerol.

[0044] Figure 9 For the multifunctional properties of the collagen-based hydrogel designed with high light transmittance and excellent mechanical strength in Example 3, where Figure A shows a picture display of the shape plasticity of the collagen-based hydrogel, and Figure B shows a picture display of the freeze-dried and rehydrated ready-to-use collagen-based hydrogel. Detailed implementation manners

[0045] In order to clearly and completely describe the technical solutions of the embodiments of the present invention in conjunction with the accompanying drawings, it is obvious that the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the present invention.

[0046] In the following embodiments, all type I collagen involved is extracted from the cowhide of calves. It has been freeze-dried before use. The collagen sponge is directly dissolved in an acetic acid solution of a certain concentration, and a fixed concentration needs to be maintained each time it is used.

[0047] Example 1

[0048] The collagen-based hydrogel with high light transmittance and excellent mechanical properties for corneal injury repair provided in this embodiment is prepared according to the following steps:

[0049] (1) Prepare a thermodynamically self-assembled hydrogel (Col)

[0050] Dissolve type I collagen from fetal bovine skin in a 0.5 M acetic acid solution at a concentration of 10 mg / ml. After complete dissolution, adjust the pH to 7.4 at 4 °C using a 5 M sodium hydroxide solution. Then centrifuge at 6000 r / min for 1 minute at 4 °C to remove internal air bubbles. After taking it out, transfer the collagen solution to a ring made of polytetrafluoroethylene using a syringe and place it in a 37 °C constant temperature incubator. After incubating for 30 min, remove the ring to obtain a thermodynamically self-assembled collagen hydrogel (Col)

[0051] (2) Prepare a collagen hydrogel with high light transmittance (Col-Gly)

[0052] Soak the collagen hydrogel (Col) obtained in step (1) in a glycerol solution, where the volume ratio of glycerol to water is 4:1, and the treatment time is 30 min. After soaking is completed, quickly rinse the residual solution on the surface with UP water to obtain a highly transparent collagen-based hydrogel, named Col-Gly.

[0053] (3) Prepare a collagen-based hydrogel (Col-Gly-OX) with both high light transmittance and excellent mechanical properties

[0054] Soak the high-light-transmittance collagen-based hydrogel (Col-Gly) obtained in step (2) in a mixed solution composed of glycerol, oxazolidine, and water. The volume of the polar molecular solution is kept at 50%, and the concentration of the cross-linking agent is kept at 10%. After soaking for 20 min, take it out and quickly rinse the residual solution on the surface with UP water to obtain a collagen-based hydrogel (Col-Gly-OX) with both high light transmittance and excellent mechanical properties.

[0055] Example 2

[0056] The preparation method of the collagen-based hydrogel with high light transmittance and excellent mechanical properties for corneal injury repair provided in this example is as follows:

[0057] (1) Prepare a thermodynamically self-assembled hydrogel (Col)

[0058] Dissolve type I collagen from fetal bovine skin in a 0.5 M glacial acetic acid solution at a concentration of 10 mg / ml. After complete dissolution, adjust the pH to 7.4 at 4°C using a 5 M sodium hydroxide solution. Then centrifuge at 6000 r / min for 1 minute at 4°C to remove internal air bubbles. After taking it out, transfer the collagen solution to a ring made of polytetrafluoroethylene using a syringe and place it in a constant temperature incubator at 37°C. After incubating for 30 min, remove the ring to obtain a thermodynamically self-assembled collagen hydrogel (Col).

[0059] (2) Prepare a high-light-transmittance collagen-based hydrogel (Col-Gly)

[0060] Soak the collagen hydrogel (Col) obtained in step (1) in a glycerol solution, where the volume ratio of glycerol to water is 3:2, and the treatment time is 30 min. After soaking is completed, quickly rinse the residual solution on the surface with UP water to obtain a highly transparent collagen-based hydrogel, named Col-Gly.

[0061] (3) Prepare a collagen-based hydrogel (Col-Gly-GA) with both high light transmittance and excellent mechanical properties

[0062] Immerse the highly transparent collagen-based hydrogel (Col-Gly) obtained in step (2) in a mixed solution composed of glycerol, oxazolidine, and water. The volume of the polar molecular solution is maintained at 50%, and the concentration of the cross-linking agent is maintained at 10%. After soaking for 20 minutes, take it out and quickly rinse the residual solution on the surface with up water to obtain a collagen-based hydrogel (Col-Gly-OX) that combines high transparency and excellent mechanical properties.

[0063] Example 3

[0064] The preparation method of the collagen-based hydrogel with high transparency and excellent mechanical properties for corneal injury repair provided in this example is as follows:

[0065] (1) Prepare a thermodynamically self-assembled hydrogel (Col)

[0066] Dissolve type I collagen from fetal bovine skin in a 0.5 M glacial acetic acid solution at a concentration of 10 mg / ml. After complete dissolution, adjust the pH to 7.4 with a 5 M sodium hydroxide solution at 4°C. Then centrifuge at 6000 r / min for 1 minute at 4°C to remove internal air bubbles. After taking it out, transfer the collagen solution to a ring made of polytetrafluoroethylene using a syringe and place it in a constant temperature incubator at 37°C. After incubating for 30 minutes, remove the ring to obtain a thermodynamically self-assembled collagen hydrogel (Col)

[0067] (2) Prepare a highly transparent collagen-based hydrogel (Col-Gly)

[0068] Immerse the collagen hydrogel (Col) obtained in step (1) in a glycerol solution, where the volume ratio of glycerol to water is 2:3, and the treatment time is 30 minutes. After soaking, quickly rinse the residual solution on the surface with up water to obtain a highly transparent collagen-based hydrogel, named Col-Gly.

[0069] (3) Prepare a collagen-based hydrogel (Col-Gly-OX) that combines high transparency and excellent mechanical properties

[0070] Immerse the highly transparent collagen-based hydrogel (Col-Gly) obtained in step (2) in a mixed solution composed of glycerol, oxazolidine, and water. The volume of the polar molecular solution is maintained at 50%, and the concentration of the cross-linking agent is maintained at 10%. After soaking for 20 minutes, take it out and quickly rinse the residual solution on the surface with up water to obtain a collagen-based hydrogel (Col-Gly-OX) that combines high transparency and excellent mechanical properties.

[0071] Example 4

[0072] The collagen-based hydrogel with high light transmittance and excellent mechanical properties for corneal injury repair provided by this embodiment has the following preparation method steps:

[0073] (1) Prepare a thermodynamically self-assembled hydrogel (Col)

[0074] Dissolve type I collagen derived from fetal bovine skin in a 0.5 M glacial acetic acid solution at a concentration of 10 mg / ml. After complete dissolution, adjust the pH to 7.4 at 4 °C using a 5 M sodium hydroxide solution. Then centrifuge at 6000 r / min for 1 minute at 4 °C to remove internal bubbles. After taking it out, transfer the collagen solution to a ring made of polytetrafluoroethylene using a syringe and place it in an incubator at 37 °C. After incubating for 30 min, remove the ring to obtain a thermodynamically self-assembled collagen hydrogel (Col)

[0075] (2) Prepare a collagen hydrogel with high light transmittance (Col-Gly)

[0076] Immerse the collagen hydrogel (Col) obtained in step (1) in a glycerol solution, where the volume ratio of glycerol to water is 1:4, and the treatment time is 30 min. After the immersion is completed, quickly rinse the surface residual solution with ultrapure water to obtain a highly transparent collagen-based hydrogel, named Col-Gly.

[0077] (3) Prepare a collagen-based hydrogel with both high light transmittance and excellent mechanics (Col-Gly-OX)

[0078] Immerse the high-light transmittance collagen-based hydrogel (Col-Gly) obtained in step (2) in a mixed solution composed of glycerol, oxazolidine, and water. The volume of the polar molecular solution is kept at 50%, and the concentration of the cross-linking agent is kept at 10%. After taking it out after 20 min of immersion, quickly rinse the surface residual solution with ultrapure water to obtain a collagen-based hydrogel (Col-Gly-OX) with both high light transmittance and excellent mechanics.

[0079] Example 5

[0080] The collagen-based hydrogel with high light transmittance and excellent mechanical properties for corneal injury repair provided by this embodiment has the following preparation method steps:

[0081] (1) Prepare a thermodynamically self-assembled hydrogel (Col)

[0082] Dissolve type I collagen derived from fetal bovine skin in a 0.5 M glacial acetic acid solution at a concentration of 10 mg / ml. After complete dissolution, adjust the pH to 7.4 with a 5 M sodium hydroxide solution at 4°C. Then centrifuge at 6000 r / min for 1 minute at 4°C using a high-speed centrifuge to remove internal air bubbles. After taking it out, transfer the collagen solution to a ring made of polytetrafluoroethylene using a syringe and place it in a constant temperature incubator at 37°C. After incubating for 30 min, remove the ring to obtain a thermodynamically self-assembled collagen hydrogel (Col).

[0083] (2) Preparation of a highly transparent collagen hydrogel (Col-Gly)

[0084] Immerse the collagen hydrogel (Col) obtained in step (1) in a glycerol solution, where the volume ratio of glycerol to water is 4:1, and the treatment time is 30 min. After the immersion is completed, quickly rinse the residual solution on the surface with up water to obtain a highly transparent collagen-based hydrogel, named Col-Gly.

[0085] (3) Preparation of a collagen-based hydrogel (Col-Gly-OX) with both high transparency and excellent mechanical properties

[0086] Immerse the highly transparent collagen-based hydrogel (Col-Gly) obtained in step (2) in a mixed solution composed of glycerol, oxazolidine, and water. The volume of the polar molecular solution is kept at 50%, and the concentration of the cross-linking agent is kept at 5%. After taking it out after 20 min of immersion, quickly rinse the residual solution on the surface with up water to obtain a collagen-based hydrogel (Col-Gly-OX) with both high transparency and excellent mechanical properties.

[0087] Example 6

[0088] The preparation method of the collagen-based hydrogel with high transparency and excellent mechanical properties for corneal injury repair provided in this example is as follows:

[0089] (1) Preparation of a thermodynamically self-assembled hydrogel (Col)

[0090] Dissolve type I collagen derived from fetal bovine skin in a 0.5 M glacial acetic acid solution at a concentration of 10 mg / ml. After complete dissolution, adjust the pH to 7.4 with a 5 M sodium hydroxide solution at 4°C. Then centrifuge at 6000 r / min for 1 minute at 4°C using a high-speed centrifuge to remove internal air bubbles. After taking it out, transfer the collagen solution to a ring made of polytetrafluoroethylene using a syringe and place it in a constant temperature incubator at 37°C. After incubating for 30 min, remove the ring to obtain a thermodynamically self-assembled collagen hydrogel (Col).

[0091] (2) Preparation of Collagen Hydrogel with High Transparency (Col-Gly)

[0092] Immerse the collagen hydrogel (Col) obtained in step (1) in a glycerol solution, where the volume ratio of glycerol to water is 4:1, and the treatment time is 30 min. After the immersion is completed, quickly rinse the residual solution on the surface with ultrapure water to obtain a highly transparent collagen-based hydrogel, named Col-Gly.

[0093] (3) Preparation of Collagen-Based Hydrogel with Both High Transparency and Excellent Mechanics (Col-Gly-OX)

[0094] Immerse the collagen-based hydrogel with high transparency (Col-Gly) obtained in step (2) in a mixed solution composed of glycerol, oxazolidine, and water. The volume of the polar molecular solution is kept at 50%, and the concentration of the cross-linking agent is kept at 2%. After taking it out after soaking for 20 min, quickly rinse the residual solution on the surface with ultrapure water to obtain a collagen-based hydrogel (Col-Gly-OX) that combines high transparency and excellent mechanics.

[0095] The present invention provides a simple method for preparing a collagen hydrogel with high light transmittance and excellent mechanical properties. First, an opaque collagen gel is prepared by the thermodynamic self-assembly method of collagen fibers. The specific process is to dissolve type I collagen derived from fetal bovine skin in an acetic acid solution. After complete dissolution, adjust the pH to 7.4 with a sodium hydroxide solution at 4°C. Then, centrifuge at high speed to remove internal air bubbles. After taking it out, transfer it to a ring made of polytetrafluoroethylene and place it in a constant temperature incubator at 37°C. After the self-assembly is completed, a thermodynamically self-assembled collagen hydrogel (Col) is obtained. To better control the properties of the collagen hydrogel, the concentration and self-assembly time used in the preparation of the thermodynamically self-assembled collagen hydrogel in the present invention are kept constant.

[0096] Example 7 Concentration Screening Test of Polar Molecular Glycerol Solution in the Present Invention

[0097] In the experiment to explore the light transmittance of the glycerol solution on the collagen hydrogel, a series of control experiments were carried out. The results showed that the improvement of the light transmittance of the collagen hydrogel has a certain concentration and time dependence. Specifically, a low-concentration glycerol solution (below 20%) does not affect the light transmittance of the collagen hydrogel. As the glycerol concentration increases, the light transmittance of the collagen hydrogel has a certain time dependence. The possible reason is that the increase in glycerol concentration will accelerate its diffusion rate into the gel, resulting in a gradual increase in its light transmittance over time. In addition, due to the certain viscosity of glycerol, when the concentration is 100%, it will limit its molecular movement rate to a certain extent, resulting in a slower increase in light transmittance than the 80% ratio group. Therefore, 4:1 is selected as the best preferred group (see Figure 1)。

[0098] Other polar molecules such as urea can also make the collagen hydrogel transparent. However, this treatment method will, on the one hand, denature the collagen gel, and on the other hand, destroy the structure and mechanical strength of the gel. It has been found that glycerol is the best polar molecule currently found that can efficiently make the collagen gel transparent.

[0099] Example 8 Screening test of the crosslinking agent oxazolidine of the present invention

[0100] Col-Gly is derived from the collagen gel treated with glycerol solution, and its properties mainly come from the concentration of the initial collagen in the collagen gel. The concentration range selected in the experiments of the present invention is 5-15 mg / ml. The higher the concentration, the more stable the properties of the Col-Gly gel. Therefore, the optimal concentration ratio of the Col-Gly gel in the present invention is 15 mg / ml.

[0101] Polar molecules have an optimal ratio. Since a low-concentration polar molecule glycerol solution cannot make the collagen gel transparent, when the transparent collagen gel is placed in a low-concentration polar molecule glycerol solution, the light transmittance of the collagen gel will decrease due to the outward diffusion of the glycerol solution inside the gel. Therefore, a high-concentration polar molecule solution (i.e., glycerol solution) is an important factor for the stable transparent state of the collagen gel. The concentrations that can be considered in the present invention are 60%, 80%, and 100%. Considering the concentration of glycerol used to make the collagen gel transparent by glycerol treatment, the optimal concentration in the step of preparing Col-Gly-OX is also 80%.

[0102] Other crosslinking agents can also achieve the maintenance of the light transmittance of the collagen gel and the improvement of its mechanical properties. Glutaraldehyde (GA) is one of them. However, as the data in the present invention has mentioned, although the glutaraldehyde treatment group (Col-Gly-GA) can improve the mechanical strength of the collagen gel, its elongation at break is lower and it cannot meet the requirements of corneal suture. Therefore, the comprehensive performance of the oxazolidine crosslinking group (Col-Gly-OX) is better.

[0103] The preparation process and properties of the collagen-based hydrogel in Examples 1-6 are analyzed below

[0104] Test Example 1 Testing the light transmittance and mechanical properties of the gel of the present invention

[0105] I. Testing the light transmittance of the gel of the present invention

[0106] Experimental materials: Collagen gel (Col) completed by self-assembly, polar molecule glycerol solution.

[0107] Experimental method: Direct observation and testing the change of the light transmittance of the collagen gel using an ultraviolet-visible light spectrometer.

[0108] Experimental results: First, the light transmittance of the opaque collagen hydrogel (Col) treated with glycerol solution was measured. As Figure 2 shown in A, as the treatment time extended, the light transmittance of the collagen hydrogel gradually increased, and the stamens of the flower below could be clearly seen. By measuring the film transmittance of the collagen gel through UV-Vis absorption spectrum (as Figure 2 shown in B), short-term immersion in glycerol (about 15 min) could make the transmittance of collagen reach over 95%. This improvement in light transmittance meets the usage requirements of corneal transplantation materials and will greatly increase its application in the field of corneal injury repair.

[0109] II. Morphology test of the collagen gel with improved light transmittance of the present invention:

[0110] Experimental materials: Self-assembled collagen gel (Col), and self-assembled collagen gel treated with glycerol (Col-Gly). Experimental methods: TEM test, AFM test, CLSM test.

[0111] Experimental results:

[0112] As Figure 3 shown in A, the fiber size of the thermodynamically self-assembled collagen gel was about 100 nm, and the fibers were arranged disorderly. Atomic force microscope (AFM) also observed thick fibers. Under the reflection mode of confocal laser scanning microscope (CLSM), collagen fibers could be observed, and it was also found that the simple thermodynamically self-assembled hydrogel had thick and disorderly fiber arrangements. While Figure 3 B shows that after treatment with glycerol, the fiber size of the collagen gel decreased significantly, about 10 nm, and only fine fibers were observed under AFM and CLSM. This decrease in fiber size will reduce light scattering and increase the light transmittance of the gel.

[0113] III. Mechanism test of the improved light transmittance of the gel of the present invention:

[0114] Experimental materials: Self-assembled collagen gel treated with glycerol (Col-Gly).

[0115] Experimental methods: Polarizing light microscope observation, two-dimensional small-angle scattering experiment, circular dichroism experiment, and infrared test.

[0116] Experimental results: As Figure 4As shown in the figure, the change of the crystalline domain during the process of treating collagen gel with glycerol was observed by polarized light microscopy. It was found that with the extension of the treatment time, the crystalline area between collagen fibers gradually decreased, and the treatment with glycerol on the surface caused a change in the crystallinity of the collagen hydrogel. Further, small-angle X-ray scattering in two dimensions found that the diffraction rings of the collagen gel after glycerol treatment decreased, indicating a reduction in the crystalline domain of Col-Gly and a decrease in the crystal size. Statistical data also showed that the diffraction peak of Col-Gly was sharpened, indicating that the arrangement of collagen fibers became more orderly.

[0117] The stability of the triple helix structure of collagen is of great significance for maintaining the biological activity of collagen hydrogel. Therefore, the structures of Col and Col-Gly in Example 3 were characterized by infrared spectra and circular dichroism spectra. The results are as Figure 5 shown. It was found that the treatment with glycerol did not change the characteristic absorption peaks of collagen fibers, so the triple helix structure remained stable.

[0118] IV. Chemical cross-linking reaction formula of the collagen gel in the present invention:

[0119] Although the collagen gel treated with pure glycerol has excellent light transmittance, this light transmittance is unstable. When placed in water, the light transmittance will decrease. The reason for this change may be that glycerol leaks in water, causing the reassembly of fibers, so the strength will decrease. In addition, the treatment of the collagen gel with pure glycerol will also cause a decrease in the mechanical strength of the collagen gel. Therefore, the present invention chemically cross-links Col-Gly in Example 1. The chemical reaction principle is as Figure 6 shown. Glutaraldehyde undergoes a Schiff base reaction with collagen amino groups through aldehyde groups to increase cross-linking. And oxazolidine can combine with collagen amino groups through covalent and non-covalent reactions through ring-opening reactions.

[0120] The present invention conducts optical tests on the chemically cross-linked collagen gel. Experimental materials: Collagen gel cross-linked with glutaraldehyde, collagen gel cross-linked with oxazolidine. Experimental method: Ultraviolet-visible light absorption method. Experimental results: The results are as Figure 7 shown. In the visible light range (380nm - 800nm), the collagen hydrogel cross-linked with oxazolidine has a light transmittance as high as 90%, and the light transmittance is about 92% at 500nm. The haze value test also found that the collagen-based hydrogel cross-linked with oxazolidine has a low haze value, meeting the requirements for corneal transplantation.

[0121] V. The present invention conducts mechanical property tests on the chemically cross-linked collagen gel.

[0122] Experimental materials: Collagen gel cross-linked with glutaraldehyde, collagen gel cross-linked with oxazolidine.

[0123] Experimental method: Stress-strain test.

[0124] Experimental results: As an artificial cornea substitute, having a modulus that conforms to corneal tissue and the suture strength required for surgical suturing are important conditions for corneal transplantation. Traditional glutaraldehyde-crosslinked collagen gels are more brittle, can be easily broken, and are difficult to meet the requirements of corneal suturing. Oxazolidine, as a new type of crosslinking agent, can bind to collagen fibers through covalent-non-covalent methods, so it can endow collagen hydrogels with excellent mechanical properties. As Figure 7 shown in A, the stress-strain curve shows that although Col-Gly-GA has a high stress, its elongation at break is only 20%, indicating a large brittleness. The stress value of the Col-Gly-OX group is relatively low, but the elongation at break it can withstand is as high as 60%. And through targeted measurement of the suture strength, it is found that the Col-Gly-OX group can withstand a suture force of about 50 gf, which is an ideal material to meet the application requirements of corneal transplantation.

[0125] VI. The present invention measures the applicability of the chemically crosslinked collagen gel.

[0126] The cornea has a curvature structure, and the existence of curvature makes the cornea an important refractive tissue of the eye. The glycerol-treated collagen gel proposed by the present invention has a low mechanical strength (as Figure 8 shown), and this collagen membrane can adapt to various shaped surfaces. As Figure 9 shown in A, this collagen gel can adapt to a rod-shaped structure to construct a ring-shaped transparent membrane material. In addition, this gel can also adapt to materials with a curved base to construct a transparent membrane material with a shape similar to that of the cornea. Interestingly, after freeze-drying and rehydration, the shape of the collagen gel can be well preserved, and the surface of the oxazolidine-treated group is smoother, indicating its potential application as a corneal transplantation substitute for emergency use in a battlefield environment.

[0127] Experimental summary: The above performance analysis shows that the collagen-based artificial cornea substitute for corneal injury repair provided by the present invention is not only simple to prepare, but also has excellent optical properties (high light transmittance and low haze value), and has high mechanical properties to meet the requirements of corneal suturing. At the same time, the shape adaptability of the collagen membrane also enables it to meet the design requirements of the corneal curvature structure. In particular, its shape retention characteristics after freeze-drying and rehydration are expected to be used as a corneal transplantation substitute in emergency situations such as on the battlefield. These properties are of great significance for solving the shortage of corneal donors and developing collagen-based artificial cornea substitutes for corneal injury repair.

[0128] Those of ordinary skill in the art will realize that the embodiments described herein are provided to assist the reader in understanding the principles of the present invention, and it should be understood that the scope of protection of the present invention is not limited to such specific statements and embodiments. Those of ordinary skill in the art can make various other specific deformations and combinations that do not depart from the essence of the present invention based on these technical revelations disclosed in the present invention, and these deformations and combinations are still within the scope of protection of the present invention.

Claims

1. A collagen-based hydrogel for corneal injury repair, characterized in that: First, prepare type I collagen into a thermodynamically self-assembled collagen hydrogel Col, and soak it in a polar molecule solution to prepare a collagen-based hydrogel Col-Gly; The type I collagen described above is derived from fetal bovine skin; The volume ratio of the polar molecule solution to the collagen hydrogel Col is greater than 10; The polar molecule soaked in the polar molecule solution is glycerol, and the volume ratio of the polar molecule to water is 1:4 - 4:1; The soaking time in the polar molecule solution is 1 - 60 min; The preparation method of the thermodynamically self-assembled collagen hydrogel Col is as follows: a. Dissolve type I collagen derived from fetal bovine skin in a 0.1 - 0.5 M glacial acetic acid solution, and the concentration of type I collagen is 5 - 15 mg / ml; b. After complete dissolution of the type I collagen solution, adjust the pH to 7.1 - 8 at 2 - 4 °C using a 1 - 5 M sodium hydroxide solution; then centrifuge at 3000 - 9000 r / min at 2 - 4 °C with a high-speed centrifuge to remove internal air bubbles; c. After taking it out, transfer the collagen solution to a ring made of polytetrafluoroethylene using a syringe, and place it in a constant temperature incubator at 30 °C - 45 °C for 20 - 60 min to obtain the thermodynamically self-assembled collagen hydrogel Col; Soak the collagen-based hydrogel Col-Gly in a mixed solution composed of a polar molecule, a cross-linking agent, and water to prepare a collagen-based hydrogel Col-Gly-OX; wherein, the polar molecule is glycerol, and the cross-linking agent is oxazolidine.

2. The collagen-based hydrogel for corneal injury repair according to claim 1, wherein: The polar molecule soaked in the polar molecule solution is glycerol, and the volume ratio to water is 4:1; the soaking time in the polar molecule solution is 30 min.

3. The collagen-based hydrogel for corneal injury repair according to claim 1, characterized in that: The type I collagen is replaced with that derived from other livestock and poultry source animal tissues.

4. The collagen-based hydrogel for corneal injury repair according to claim 1, characterized in that: The temperature in the constant temperature incubator is 37 °C.

5. The collagen-based hydrogel for corneal injury repair according to claim 1, wherein: The volume of the polar molecule solution in the mixed solution is 50 - 60% or 80%, and the concentration of the cross-linking agent is 2% - 20%; the concentration range of the collagen-based hydrogel Col-Gly is 5 - 15 mg / ml.

6. The collagen-based hydrogel for corneal injury repair according to claim 5, characterized in that: The volume of the polar molecule solution in the mixed solution is 80%, and the concentration of the cross-linking agent is 10%.

7. The collagen-based hydrogel for corneal injury repair according to claim 1, wherein: The soaking time in the mixed solution composed of a polar molecule, a cross-linking agent, and water is 2 - 30 min.

8. The collagen-based hydrogel for corneal injury repair according to any one of claims 1-7, characterized in that: The hydrogel contains a compound: 。 9. Use of the collagen-based hydrogel according to any one of claims 1 - 8 in the preparation of a material for corneal injury repair.

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