A combination product for corneal lesion repair and a method for preparing the same

Abstract

The application provides a combination product for corneal lesion repair, and belongs to the technical field of biomedical materials. The combination product comprises a high-transparency collagen-based artificial cornea and an adherable gel scaffold carrying seed cells. The artificial cornea is prepared by an electrochemical deposition method, and has controllable diameter, thickness and curvature, and a light transmittance of more than 90%. The gel scaffold is composed of recombinant collagen, polyethylene glycol and seed cells. The gel precursor liquid with good fluidity can fully fill the corneal wound and in-situ deliver the seed cells, and the gel is formed and adhered in-situ through a spontaneous chemical cross-linking reaction, so that the corneal graft is suture-free transplanted. Meanwhile, the gel scaffold can be degraded according to the proliferation of the seed cells, which is beneficial to the deep repair of the corneal tissue. The combination product has the advantages of convenient operation, postoperative recovery speed and effect, and is expected to replace the current single corneal graft or corneal tissue engineering scaffold treatment method, and has a good clinical application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to a combination product for the repair of corneal lesions. Background Technology

[0002] More and more patients are experiencing corneal opacity due to corneal diseases, resulting in moderate to severe visual impairment. However, due to a severe shortage of donor corneas, only a small number of patients can undergo transplantation. In recent years, with the rise and development of artificial corneal materials and corneal tissue engineering, damaged corneas can be repaired in situ through artificial corneal transplantation or by using seed cells and scaffold materials, bringing new hope to the treatment of patients with corneal blindness.

[0003] Artificial corneal transplantation is currently the main treatment for restoring vision in patients with corneal blindness. An artificial corneal graft that replaces the diseased cornea can help patients quickly regain some vision. In China, acellular porcine stromal grafts are the mainstream clinical application. However, such materials inevitably carry risks such as residual antigens and viruses. After implantation, a high rejection rate can lead to corneal opacity, and the curvature and thickness are uncontrollable, making personalized customization impossible. Furthermore, current corneal transplant surgeries connect the corneal graft to the damaged corneal stroma using sutures. However, suturing, as a connection method, is not only time-consuming and requires a high level of skill, but it is also associated with many potential complications after corneal transplantation: ① Sutures may cause further damage to the cornea, leading to inflammation, scarring, and neovascularization, thus affecting corneal transparency; ② The suture itself, as a foreign body, may cause rejection, inducing inflammation, and even leading to transplant failure; ③ Sutures cannot guarantee sufficient contact between the implant and the damaged stroma layer, potentially resulting in poor wound adhesion or surface distortion, thereby impairing visual quality. Therefore, there is an urgent need to develop a personalized artificial cornea that combines high transparency and high biocompatibility, while introducing sutureless technology to achieve "seamless" transplantation for corneal patients.

[0004] Patent (CN114618016A) discloses an artificial cornea and its preparation method. The method includes designing a customized electrode, assembling collagen using improved EDP technology, and chemical cross-linking steps to obtain a collagen-based artificial cornea with a shape consistent with the cornea on the electrode. The collagen-based artificial cornea material is characterized by being composed of short-range ordered and densely arranged collagen microfibers, having a transparency of over 80%, controllable thickness, and controllable curvature. This invention does not require complex equipment, and the obtained collagen-based artificial cornea material can be used to replace or repair the natural cornea.

[0005] Patent (CN113637067A) provides a recombinant human collagen and its artificial cornea, which is prepared from recombinant collagen and chitosan. The artificial cornea process of this invention does not require harsh conditions or unsafe raw materials and reagents, has good biocompatibility, moderate mechanical strength, is suitable for use in cornea, has high light transmittance, good physicochemical properties, is free of impurities, and has good shapeability. It can be used in corneal replacement, corneal repair scaffolds, suitable corneal contact lenses, in vitro culture of corneal cells, and ocular drug sustained-release carriers, and other related fields.

[0006] Corneal tissue engineering, as an emerging treatment method, repairs damaged corneal tissue by implanting scaffold materials carrying active functional seed cells. Currently, commonly used scaffold materials are mainly decellularized matrix tissue and hydrogel. However, due to limitations in raw material composition or design concepts, existing scaffold materials still have the following problems: ① They cannot simulate the shape, thickness, or curvature of the natural cornea, which is a crucial factor affecting corneal refractive power and has a decisive impact on vision recovery; ② The degradation rate of the material is uncontrollable and cannot match the proliferation rate of the carried cells or the repair rate of the damaged tissue, resulting in poor treatment outcomes; ③ They generally lack tissue adhesion, failing to adhere tightly to the damaged corneal stroma, leading to slow graft integration, hindering the repair of deep lamellar corneal tissue, and easily causing astigmatism, thus affecting treatment effectiveness.

[0007] Therefore, how to utilize artificial corneas and corneal tissue engineering scaffolds, leveraging their complementary advantages, to help corneal blind patients, especially those with deep lamellar keratitis, to quickly regain some vision in the early stages of treatment while simultaneously achieving deep repair of corneal tissue in the later stages, has significant research and application value. Summary of the Invention

[0008] This invention addresses the problems of existing technologies by providing a combined product for corneal lesion repair. The combined product comprises a highly transparent collagen-based artificial cornea and an adhesive gel scaffold carrying seed cells. The artificial cornea is fabricated using an electrochemical deposition method, with controllable diameter, thickness, and curvature, achieving a light transmittance of over 90%. The gel scaffold is composed of recombinant collagen, polyethylene glycol, and seed cells. A highly fluid gel precursor solution effectively fills the corneal wound and delivers seed cells in situ. These cells then undergo a spontaneous chemical cross-linking reaction to form a gel in situ and adhere, enabling sutureless corneal graft transplantation. Simultaneously, the gel scaffold degrades as the seed cells proliferate, facilitating deep corneal tissue repair. This combined product balances surgical convenience with postoperative recovery speed and effectiveness, and is expected to replace current single-method treatments such as corneal grafts or corneal tissue-engineered scaffolds, demonstrating excellent clinical application prospects.

[0009] To achieve the above objectives, in a first aspect, the present invention provides a combination product for the repair of corneal lesions, the combination product comprising: a highly transparent collagen-based artificial cornea and an adhesive gel scaffold carrying seed cells.

[0010] Preferably, the highly transparent collagen-based artificial cornea is prepared by electrochemical deposition; the raw material used in the highly transparent collagen-based artificial cornea is type I collagen. The light transmittance of the highly transparent collagen-based artificial cornea is above 90%, and its diameter, curvature, thickness, etc., can be customized by changing the electrodeposition parameters or the shape of the working electrode.

[0011] Preferably, the adhesive gel scaffold is composed of recombinant collagen peptides, active esterified polyethylene glycol, and seed cells; the recombinant collagen peptides are obtained by gene fermentation technology and include one or two of recombinant type I human collagen and recombinant type III human collagen, more preferably recombinant type III human collagen; the active esterified polyethylene glycol is selected from polyethylene glycol succinimide succinate and / or polyethylene glycol succinimide glutarate, more preferably the active esterified polyethylene glycol is a combination of polyethylene glycol succinimide glutarate (PEG-SG) and succinimide succinate (PEG-SS), and the mass ratio of PEG-SG to PEG-SS is (1-10):1.

[0012] Preferably, the polyethylene glycol structure in the active esterified polyethylene glycol includes one or more of linear double-ended polyethylene glycol, three-armed polyethylene glycol, four-armed polyethylene glycol, six-armed polyethylene glycol, and eight-armed polyethylene glycol, more preferably four-armed polyethylene glycol. The molecular weight of the active esterified polyethylene glycol is preferably 5-40 kDa.

[0013] Preferably, the seed cells are selected from one or more of corneal endothelial cells, mesenchymal stem cells, and limbal stem cells.

[0014] Preferably, the seed cells are derived from one or more of the following: primary cell culture, cell passage culture, and resuscitation of frozen cells.

[0015] Preferably, the seed cells are suspended in the precursor solution of the gel and delivered in situ to the damaged corneal area.

[0016] Secondly, the present invention provides a method for preparing a combination product for corneal lesion repair, the preparation process including: preparation of a highly transparent collagen-based artificial cornea and preparation of an adhesive gel scaffold;

[0017] The preparation of a highly transparent collagen-based artificial cornea includes the following steps:

[0018] (1) Setup of the electrodeposition apparatus: A three-electrode system for electrodeposition is adopted, with a titanium plate of a certain curvature as the working electrode, Ag / AgCl as the reference electrode, and platinum wire as the counter electrode;

[0019] (2) Preparation of collagen solution: Prepare collagen solution with acetic acid as solvent, add hydrogen peroxide to the collagen solution to a concentration of 0.5M, stir and mix well, centrifuge to remove air bubbles, and set aside for use;

[0020] (3) Electrochemical deposition process: The three electrodes in step (1) are immersed in the acidic collagen solution obtained in step (2), a constant current density is applied to the electrodes, and after electrodeposition for a period of time, a highly transparent collagen cornea with a certain thickness is obtained on the surface of the working electrode.

[0021] (4) Crosslinking fixation: The cornea obtained in step (3) is irradiated under 265nm ultraviolet light for 1-4 hours.

[0022] Preferably, in step (1), the curvature of the titanium plate is 7.8-8.4, and the three-electrode system is CHI660E; in step (2), the concentration of the acetic acid is 1-2%; the concentration of the collagen solution is 0.5-1% w / v; and in step (3), the electrodeposition time is 10-30 min, and the current density is 3.33 mA / cm². 2 Step (4) Irradiate for 2 hours.

[0023] The preparation of the adhesive gel scaffold includes the following steps:

[0024] (1) Add phosphate buffer dropwise to the sample well containing recombinant collagen lyophilized powder, and stir evenly with a stirring rod to obtain recombinant collagen solution;

[0025] (2) Use a dropper to draw the recombinant collagen solution from step (1) into a test tube containing seed cells, and slowly blow it to suspend the cells and obtain a cell suspension.

[0026] (3) Add physiological saline to the sample trough containing active esterified polyethylene glycol, stir evenly with a stirring rod, and use a dropper to draw the polyethylene glycol solution into the test tube of step (2), and slowly blow it evenly to obtain the solution.

[0027] Preferably, the recombinant collagen solution in step (1) has a mass-volume concentration of 5-20% w / v; the cell suspension in step (2) has a cell concentration of 10%. 4 Up to 10 6 Cells per milliliter;

[0028] Thirdly, the present invention provides the application of the above-mentioned combined product in the preparation of corneal lesion repair products.

[0029] This invention is applicable to patients with infectious keratitis whose corneal structure has been irreversibly damaged, resulting in decreased or lost vision, and who require corneal transplantation. A highly transparent collagen-based artificial cornea is used as an implant to replace or repair the diseased cornea, and limbal stem cells are delivered in situ using a gel scaffold. Simultaneously, the corneal graft and damaged corneal tissue are tightly bonded, achieving sutureless transplantation and promoting deep corneal repair starting from the endothelium.

[0030] The beneficial effects of this invention are:

[0031] ① Compared to treatment methods that use a single corneal tissue engineering scaffold or corneal graft, the combined product in this invention uses a customizable corneal graft to replace the diseased corneal tissue, helping patients quickly regain some vision. At the same time, the gel scaffold can promote the repair of deep corneal tissue and improve the visual quality in the later stages. With the complementary advantages of both, the postoperative repair effect can be effectively improved, providing a new treatment method for patients with corneal blindness.

[0032] ② Compared with traditional decellularized matrix tissue engineering scaffolds, the gel scaffold in this invention uses synthetic raw materials as the gelling component, eliminating the risk of immunogenicity. At the same time, the gel scaffold has the ability to gel in situ and adhere. The gel precursor solution has excellent fluidity, which can fully fill the defect site, ensure full contact between the corneal graft and the damaged matrix layer, and achieve sutureless transplantation. This avoids the damage risks and possible series of complications caused by suture operations, effectively reduces the formation of scars, inflammation and new blood vessels, reduces the difficulty of surgical operation, and saves surgical time.

[0033] ③ Compared with traditional hydrogel tissue engineering scaffolds, the gel scaffold in this invention uses highly bioactive recombinant collagen and specially designed polyethylene glycol derivatives as gelling components. While having good cell adhesion, it also has a controllable degradation rate, which can degrade as needed along with cell proliferation, providing sufficient growth space for them, thereby improving the speed and quality of tissue repair.

[0034] ④ Compared to the mainstream decellularized stromal grafts of porcine cornea, the artificial cornea in this invention is made from type I collagen through electrochemical deposition, which has a short preparation cycle and does not require the use of cross-linking agents or other additives. At the same time, the artificial cornea in this product can be transplanted without sutures through a gel scaffold, so weak cross-linking can be used, sacrificing some mechanical properties in exchange for higher transparency, bioactivity and safety, thereby improving the repair effect. Attached Figure Description

[0035] Figure 1 Transmittance testing of collagen artificial corneas and gel scaffolds in Examples 1 and 2.

[0036] Figure 2Fluorescent staining image of limbal stem cells on the surface of the gel scaffold in group B of Example 2.

[0037] Figure 3 The proliferation of limbal stem cells on the surface of the gel scaffold in group B in Example 2. Detailed Implementation

[0038] It is worth noting that the raw materials used in this invention are all commercially available products, and their sources are not specifically limited. The invention will now be described in detail with reference to specific embodiments.

[0039] Example 1:

[0040] In this embodiment, a method for preparing a combination product for corneal lesion repair is provided, as follows:

[0041] Preparation of highly transparent collagen-based artificial corneas:

[0042] (1) Setup of the electrodeposition apparatus: A three-electrode system (CHI660E) for electrodeposition was adopted, with a titanium plate of a certain curvature as the working electrode, Ag / AgCl as the reference electrode, and platinum wire as the counter electrode.

[0043] (2) Preparation of collagen solution: Prepare a 0.5% (w / v) collagen solution using 0.5% acetic acid as solvent, and add hydrogen peroxide to the collagen solution to a concentration of 0.5M. Stir and mix well, then centrifuge to remove air bubbles and set aside for use.

[0044] (3) Electrochemical deposition process: The three electrodes described above are immersed in the acidic collagen solution described above, and a constant current density (3.33 mA / cm²) is applied to the electrodes. 2 After electrodeposition for 10 minutes, a highly transparent collagen cornea with a certain thickness was obtained on the surface of the working electrode.

[0045] (4) Crosslinking fixation: The cornea obtained in step (3) was irradiated under 265nm ultraviolet light for 120min.

[0046] Preparation of adhesive gel scaffolds:

[0047] (1) Add 5 mL of phosphate buffer to the sample well containing 0.4 g of recombinant type III human collagen lyophilized powder and stir evenly with a stir bar;

[0048] (2) Use a dropper to draw up the recombinant collagen solution from step (1) into the container of limbal stem cells (cell concentration of 2×10⁻⁶). 5 The cells were suspended in a test tube containing (number / mL) and slowly pipetted.

[0049] (3) Add 5 mL of physiological saline to the sample well containing 0.4 g of active esterified polyethylene glycol (four-arm polyethylene glycol succinimide glutarate 4-arm-PEG-SG and four-arm succinimide succinimide 4-arm-PEG-SS), stir evenly with a stirring rod, and use a dropper to draw the polyethylene glycol solution into the test tube of step (2), and slowly blow it evenly to obtain the solution.

[0050] This embodiment contains multiple examples, and the components of the gel scaffold in each example are shown in Table 1 below:

[0051] Table 1

[0052] 4-arm-PEG-SG (10kDa) 4-arm-PEG-SS (10kDa) Recombinant Type III Human Collagen A 0.36g 0.04g 0.4g B 0.28g 0.2g 0.4g C 0.2g 0.2g 0.4g

[0053] Example 2

[0054] In this embodiment, a method for preparing a combination product for corneal lesion repair is provided, as follows:

[0055] Preparation of highly transparent collagen-based artificial corneas:

[0056] (1) Setup of the electrodeposition apparatus: A three-electrode system (CHI660E) for electrodeposition was adopted, with a titanium plate of a certain curvature as the working electrode, Ag / AgCl as the reference electrode, and platinum wire as the counter electrode.

[0057] (2) Preparation of collagen solution: Prepare a 1% (w / v) collagen solution using 1% acetic acid as solvent, and add hydrogen peroxide to the collagen solution to a concentration of 0.5M. Stir and mix well, then centrifuge to remove air bubbles and set aside for use.

[0058] (3) Electrochemical deposition process: The three electrodes described above are immersed in the acidic collagen solution described above, and a constant current density (3.33 mA / cm²) is applied to the electrodes. 2 After electrodeposition for 20 minutes, a highly transparent collagen cornea with a certain thickness was obtained on the surface of the working electrode.

[0059] Preparation of adhesive gel scaffolds:

[0060] (1) Add 5 mL of phosphate buffer to the sample well containing 0.4 g of recombinant type III human collagen lyophilized powder and stir evenly with a stir bar;

[0061] (2) Use a dropper to draw up the recombinant collagen solution from step (1) into the container of limbal stem cells (cell concentration of 10). 6 The cells were suspended in a test tube containing (number / mL) and slowly pipetted.

[0062] (3) Add 5 mL of physiological saline to the sample well containing 0.4 g of active esterified polyethylene glycol (four-arm polyethylene glycol succinimide glutarate 4-arm-PEG-SG and four-arm succinimide succinimide 4-arm-PEG-SS), stir evenly with a stirring rod, and use a dropper to draw the polyethylene glycol solution into the test tube of step (2), and slowly blow it evenly to obtain the solution.

[0063] This embodiment contains multiple examples, and the components of the gel scaffold in each example are shown in Table 2:

[0064] Table 2

[0065] 4-arm-PEG-SG (10kDa) 4-arm-PEG-SS (20kDa) Recombinant Type III Human Collagen A 0.36g 0.04g 0.4g B 0.28g 0.12g 0.4g C 0.2g 0.2g 0.4g

[0066] Example 3: Transmittance Test of Collagen Artificial Cornea and Gel Scaffold

[0067] The collagen artificial corneas and gel scaffolds prepared in Examples 1 and 2 were soaked in ultrapure water for 1 hour to reach saturation. They were then cut into square pieces of a fixed size and placed in cuvettes. The transmittance (%) in the visible light wavelength range (380 nm to 800 nm) was measured using a UV-Vis spectrophotometer. The results are shown in the appendix. Figure 1 .

[0068] As attached Figure 1 As shown, the collagen cornea prepared by electrochemical deposition exhibits high light transmittance, exceeding 90% at wavelengths above 500 nm, which is close to that of natural corneas. The light transmittance in Example 1 is slightly higher than that in Example 2, presumably due to the lower collagen concentration and thus higher water content, resulting in higher light transmittance. The light transmittance of the gel scaffold components in Examples 1 and 2 is similar, therefore only one curve is shown in the figure.

[0069] Example 4: Gel scaffold gelation time test

[0070] The gelation time was determined using the inverted vial method. In Examples 1 and 2, the precursor solutions were uniformly mixed according to the above steps, transferred to transparent glass bottles, and placed in a 37°C constant temperature water bath. A stopwatch was used to time the gelation process, recording the time from gel formation to the point where the solution no longer flowed as the gelation time, as shown in Table 3.

[0071] Table 3

[0072]

[0073] As shown in Table 3, when the solid content and the number of active esters are fixed, the gelation time of each group is similar. The gelation time of the component ratio selected in this invention is between 2 and 3 minutes, which allows sufficient time for the bonding operation. Moreover, the precursor liquid has good fluidity before gelation, which can ensure sufficient contact with the defect area to achieve tight adhesion of the cornea.

[0074] Example 5: Adhesion performance test of gel scaffold

[0075] Adhesion strength test: After the gel scaffolds from Examples 1 and 2 were mixed evenly according to the above steps, 200 μL of solution was accurately transferred between two pieces of pigskin (1 cm × 2.5 cm) using a pipette. After complete reaction, the pigskins were fixed to one end of a roughened glass slide (5 cm × 2.5 cm) using cyanoacrylate adhesive (Golden Elephant 508). The bonded pigskins were then stretched using a universal mechanical stretching machine at a stretching rate of 5 mm / min. The maximum value measured during the stretching process corresponds to the adhesion strength of the hydrogel, and the results are shown in Table 4.

[0076] Table 4

[0077]

[0078] Bursting pressure test: A bursting pressure device was prepared, which was a sealed empty box. The bottom layer was connected to an air valve, and the top layer had a circular hole with a radius of 2 mm. The hole was connected to a conduit, and a syringe was inserted under the cornea of ​​an intact pig eyeball. Fresh pig eyeballs were selected, washed, and a circular notch with a diameter of 5 mm was created at the cornea. The gel scaffolds from Examples 1 and 2 were mixed evenly according to the above steps. 200 μL of the precursor solution was accurately transferred to the corneal notch using a pipette. After gelation, the air valve was opened to allow air to ventilate until the gel was ruptured. The air pressure inside the bursting pressure device at the time of rupture was measured, which was the bursting pressure. The results are shown in Table 5.

[0079] Table 5

[0080]

[0081] As shown in Table 5, the burst pressure of the adhesive is much higher than the normal intraocular pressure range (10-21 mmHg), which can ensure a tight seal and prevent leakage during application.

[0082] Example 6: Compressive strength test of gel scaffold

[0083] After uniformly mixing the gel scaffolds from Examples 1 and 2 according to the above steps, the precursor solution was transferred to a cylindrical mold using a pipette. (h = 7.5 mm) After it has completely gelled, it is removed and subjected to compression testing under a universal mechanical analyzer at a compression rate of 1 mm / min. The compressive strength of the hydrogel is calculated using stress-strain curves, as shown in Table 6.

[0084] Table 6

[0085]

[0086] As can be seen from the above results, thanks to the stable covalent bonds formed between multi-arm polyethylene glycol and recombinant collagen and the uniform gel network, the hydrogels in all embodiments have a certain compressive strength, which can ensure that they are not damaged by compression during application.

[0087] Example 7: Swelling Degree Test of Gel Scaffold

[0088] After uniformly mixing the gel scaffolds from Examples 1 and 2 according to the above steps, the precursor solution was transferred to a 0.5 mm mold using a pipette. Once completely gelled, the mixture was removed, cut into 15 mm diameter circular slices, and weighed (recorded as W0). The gel was transferred to centrifuge tubes, physiological saline was added, and the tubes were placed in a 37°C incubator for observation. Samples were removed every 12 hours, surface moisture was blotted off with filter paper, and the samples were weighed until the mass no longer increased (recorded as W1).

[0089] Swelling rate = (W1 / W0) × 100%, the results are shown in Table 7.

[0090] Table 7

[0091]

[0092] As shown in Table 7, the hydrogels in the examples all have low swelling rates. It is speculated that this is because the three helical peptide chains on the recombinant collagen form a tight collagen fiber network through H bonds, van der Waals forces, etc., and at the same time, a tight covalent network is formed with the multi-arm polyethylene glycol derivative. The dual network structure formed successively gives the adhesive high cohesive force to resist swelling.

[0093] Example 8: In vitro degradation time test

[0094] After uniformly mixing the two-phase precursor solutions of the gel scaffolds in Examples 1 and 2 according to the above steps, the precursor solution was transferred to a 0.5 mm mold using a pipette. After it was completely gelled, it was removed, cut into circular thin slices with a diameter of 15 mm, and transferred to centrifuge tubes. Phosphate buffer (pH 7.4) was added, and the tubes were placed in a constant temperature incubator at 37°C for observation. The time required for complete degradation was recorded, and the results are shown in Table 8.

[0095] Table 8

[0096]

[0097] Table 8 shows that the degradation time of the hydrogel can be controlled within 3-5 weeks, and the degradation rate increases with the increase of PEG-SS content. The active ester units of PEG-SS and PEG-SG are connected to the polyethylene glycol backbone through ester bonds. Hydrogel degradation occurs along with the hydrolysis of these ester bonds. By utilizing the different hydrolysis rates of PEG-SS and PEG-SG, the degradation time can be effectively controlled by adjusting the molecular weight and ratio of each component.

[0098] Example 9: Proliferation experiment of limbal stem cells on the surface of a gel scaffold

[0099] After thoroughly mixing the gel scaffold from Group B in Example 2 according to the above steps, transfer 200 μL of the precursor solution to each well of a 48-well plate using a pipette. Once gelled, limbal stem cells are then introduced at a rate of 5 × 10⁻⁶ μL. 4 Inoculate the gel surface at a density of 100 cells / mL, then add 0.5 mL of DMEM / F12 medium to each well, and incubate in a 37°C, 5% CO2 incubator, changing the medium every three days.

[0100] Observation of cell growth on the gel surface:

[0101] On days 1, 3, and 7, 1 mL of calcein-AM / PI solution was added to each well and incubated at 37°C for 20-30 min. The Calcein-AM / PI solution was then discarded, and the samples were washed with phosphate-buffered saline (PBS) and observed under a fluorescence inverted microscope. The fluorescence images were analyzed and processed using IPP 6.0 image analysis software. The results are shown in the appendix. Figure 2 Blue light excites the green fluorescence of living cells, while green light excites the red fluorescence of dead cells.

[0102] Cell proliferation assay on gel surface:

[0103] On days 1, 3, and 7, three wells were sampled, and 100 μL of 5 mg / mL MTT solution was added to each well. After incubation for 4 hours, the plates were removed, and the culture medium and MTT mixture were aspirated. The plates were then placed in a fume hood, and 200 μL of dimethyl sulfoxide solution was added to each well. After thorough shaking, the solution was transferred to a 96-well plate, and the OD values ​​of the samples were measured at 490 nm. The results are shown in the appendix. Figure 3 .

[0104] The results show that, thanks to the RGD peptides on the recombinant collagen, the hydrogel possesses excellent cell affinity, facilitating the adhesion of limbal stem cells. Simultaneously, the hydrolysis of the gel provides growth space for cell proliferation. Therefore, this gel holds promise as a cell scaffold for in-situ cell delivery and deep repair of damaged corneal tissue.

[0105] The present invention is not limited to the technical means disclosed above, but also includes technical solutions composed of any combination of the above technical features. The above descriptions are specific embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications are also considered within the scope of protection of the present invention.

Claims

1. A combination product for the repair of corneal lesions, characterized in that: The combined product comprises: a collagen-based artificial cornea and an adhesive gel scaffold carrying seed cells; wherein the collagen-based artificial cornea is prepared by electrochemical deposition; the adhesive gel scaffold is composed of recombinant collagen, active esterified polyethylene glycol, and seed cells; the polyethylene glycol structure in the active esterified polyethylene glycol includes one or more of linear double-ended polyethylene glycol, three-armed polyethylene glycol, four-armed polyethylene glycol, six-armed polyethylene glycol, and eight-armed polyethylene glycol; the active esterified polyethylene glycol is selected from polyethylene glycol succinimide succinate and / or polyethylene glycol succinimide glutarate.

2. The combined product as described in claim 1, characterized in that: The raw material used in the collagen-based artificial cornea is type I collagen.

3. The combined product as described in claim 1, characterized in that: The recombinant collagen is produced by gene fermentation technology and includes one or two of recombinant type I human collagen and recombinant type III human collagen.

4. The combined product as described in claim 3, characterized in that: The recombinant collagen is recombinant type III human collagen; the active esterified polyethylene glycol is a combination of polyethylene glycol succinimide glutarate and polyethylene glycol succinimide succinate, with a mass ratio of polyethylene glycol succinimide glutarate to polyethylene glycol succinimide succinate of (1-10):1; the polyethylene glycol in the active esterified polyethylene glycol has a four-arm polyethylene glycol structure.

5. The combined product as described in claim 1, characterized in that: The molecular weight of the active esterified polyethylene glycol is 5-40 kDa.

6. The combined product as described in claim 1, characterized in that: The seed cells are selected from one or more of corneal endothelial cells, mesenchymal stem cells, and limbal stem cells; the sources of the seed cells include one or more of primary cell culture, cell passage culture, and cryopreserved cell revival; the seed cells are suspended in the precursor solution of the gel and delivered in situ to the damaged corneal area.

7. A method for preparing the combined product as described in any one of claims 1-6, the preparation process comprising: Preparation of collagen-based artificial corneas and preparation of adhesive gel scaffolds; The preparation of collagen-based artificial corneas includes the following steps: (1) Setup of the electrodeposition apparatus: A three-electrode system for electrodeposition is adopted, with a titanium plate of a certain curvature as the working electrode, Ag / AgCl as the reference electrode, and platinum wire as the counter electrode; (2) Preparation of collagen solution: Prepare collagen solution with acetic acid as solvent, and add hydrogen peroxide to the collagen solution to a concentration of 0.5 M. Stir and mix well, then centrifuge to remove air bubbles and set aside for use. (3) Electrochemical deposition process: The three electrodes in step (1) are immersed in the acidic collagen solution obtained in step (2), a constant current density is applied to the electrodes, and after electrodeposition for a period of time, a collagen cornea with a certain thickness is obtained on the surface of the working electrode. (4) Crosslinking fixation: The cornea obtained in step (3) is irradiated under 265nm ultraviolet light for 1-4 hours; The preparation of the adhesive gel scaffold includes the following steps: (1) Add phosphate buffer dropwise to the sample well containing recombinant collagen lyophilized powder, and stir evenly with a stirring rod to obtain recombinant collagen solution; (2) Use a dropper to draw the recombinant collagen solution from step (1) into a test tube containing seed cells, and slowly blow it to suspend the cells and obtain a cell suspension. (3) Add physiological saline to the sample trough containing active esterified polyethylene glycol, stir evenly with a stirring rod, and use a dropper to draw the polyethylene glycol solution into the test tube of step (2), and slowly blow it evenly to obtain the solution.

8. The preparation method according to claim 7, characterized in that: In the preparation process of collagen-based artificial cornea, the curvature of the titanium plate in step (1) is 7.8-8.4, and the three-electrode system is CHI660E; the concentration of acetic acid in step (2) is 1-2%; the concentration of collagen solution is 0.5-1% w / v; and the electrodeposition time in step (3) is 10-30 min, with a current density of 3.33 mA / cm². 2 Step (4) Irradiate for 2 hours.

9. The preparation method according to claim 8, characterized in that: In the preparation process of the adhesive gel scaffold, the mass-volume concentration of the recombinant collagen solution in step (1) is 5-20% w / v; the cell concentration of the cell suspension in step (2) is 10%. 4 Up to 10 6 Cells per milliliter.

10. The use of the combined product as described in any one of claims 1-6 or the combined product prepared by the preparation method as described in any one of claims 7-9 in the preparation of corneal lesion repair products.

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