Oxidized dextran-modified collagen membrane for corneal repair and preparation method thereof

Abstract

This invention discloses an oxidized dextran-modified collagen membrane for corneal repair and its preparation method, relating to the field of corneal lamellar transplantation repair materials. The key technical points are: this invention selects dextran, commonly used as a plasma volume expander, from a biocompatible material, and oxidizes and grafts it to increase the active sites for cross-linking with collagen, thereby modifying collagen and improving its mechanical properties, light transmittance, and resistance to enzymatic degradation, making it better suited for corneal lamellar transplantation repair. This provides a novel, safe, and effective corneal lamellar transplantation repair material, meeting clinical treatment needs and contributing to the development of ophthalmic medical technology.

Description

Technical Field

[0001] This invention relates to the field of corneal lamellar transplantation repair materials, and more specifically, to an oxidized dextran-modified collagen membrane for corneal repair and its preparation method. Background Technology

[0002] Corneal blindness, the second leading cause of blindness worldwide, has become a serious public health problem affecting human visual health and quality of life. Statistics show that approximately 60 million people worldwide suffer from corneal blindness, and this number is increasing annually. Corneal transplantation is currently the most effective treatment for corneal blindness, but clinical practice faces an extreme shortage of corneal repair materials. Data shows that the supply-demand ratio of corneal repair materials is approximately 1:70, and this huge gap prevents many patients from receiving timely treatment, significantly impacting their quality of life.

[0003] To address this challenge, researchers have continuously explored alternative corneal repair materials. Early clinical trials attempted to use amniotic membrane, polymethyl methacrylate (PMMA), and porcine cornea. However, these materials suffer from numerous insurmountable drawbacks. While amniotic membrane, as a biological material, offers some advantages in biocompatibility, its insufficient transparency and rapid degradation in vivo prevent it from maintaining the normal morphology and function of the cornea long-term. PMMA possesses good mechanical properties, but its poor biocompatibility easily triggers immune rejection, leading to surgical failure. Although porcine cornea shares some similarities in composition with human cornea, it poses a risk of cross-species infection and has weaker mechanical properties, failing to meet the mechanical requirements of the cornea. These problems severely limited the widespread clinical application of early alternative materials.

[0004] Further research has revealed that collagen is a major component of the cornea, and its structure is similar to the natural corneal stroma, exhibiting good biocompatibility and making it an ideal candidate for lamellar corneal transplantation. However, natural collagen itself has several limitations, such as weak mechanical properties, making it difficult to support normal corneal physiological activities; easy degradation, making it unable to maintain structural stability in vivo for a long time; and low light transmittance, affecting postoperative visual quality. These shortcomings greatly limit the application of collagen in the field of lamellar corneal transplantation.

[0005] To improve the performance of collagen, researchers have explored physical modification and chemical cross-linking methods. Physical modification primarily enhances collagen's properties by altering its physical state; however, this method is inconsistent in its effectiveness and offers limited performance improvements, failing to meet clinical needs. Chemical cross-linking involves introducing chemical cross-linking agents to create cross-linked structures between collagen molecules, thereby enhancing its mechanical properties and resistance to degradation. However, these agents often possess toxicity and can remain in the material during modification, potentially triggering inflammatory reactions and immune rejection, seriously threatening patients' ocular health. This has become a key bottleneck hindering the application of collagen in corneal lamellar transplantation repair.

[0006] In summary, the field of lamellar corneal transplantation repair materials currently faces numerous challenges, including material shortages, performance defects in existing materials, and imperfect modification methods. Developing a novel, safe, and effective lamellar corneal transplantation repair material to address the shortcomings of existing materials and meet clinical treatment needs has become a crucial and urgent research topic in the field of ophthalmology. Summary of the Invention

[0007] The purpose of this invention is to provide an oxidized dextran-modified collagen membrane for corneal repair and its preparation method. Dextran, which is commonly used as a plasma volume expander, is selected from biocompatible substances and oxidized and grafted onto it to give it a certain ability to crosslink with collagen, thereby modifying collagen and improving its mechanical properties, light transmittance and resistance to enzymatic degradation, so as to better apply it to corneal lamellar transplantation repair.

[0008] The above-mentioned technical objective of the present invention is achieved through the following technical solution: a method for preparing an oxidized dextran-modified collagen membrane for corneal repair, the preparation method comprising the following steps:

[0009] S1. Preparation of oxidized dextran:

[0010] (1) Weigh out the dextran and dissolve it in PBS to prepare a dextran aqueous solution;

[0011] (2) Weigh out sodium periodate again in the dark and dissolve it in PBS to prepare a sodium periodate solution;

[0012] (3) Finally, the above solution was added to a beaker at a ratio of sodium periodate: dextran = 1:3. The mixture was stirred in the dark for 3 hours, and then a certain amount of ethylene glycol was added and stirred for another 1 hour to terminate the reaction.

[0013] (4) After the reaction is complete, the resulting solution is dialyzed for 3-4 days and then freeze-dried to obtain fluffy sponge-like oxidized dextran, which is then sealed and stored away from light;

[0014] S2. Preparation of oxidized dextran-modified collagen membrane:

[0015] (1) Weigh the oxidized dextran prepared in S1, dissolve it in deionized water to make a 0.65% oxidized dextran aqueous solution, and store it at low temperature and away from light;

[0016] (2) Weigh the freeze-dried collagen, cut it into pieces and add it to a hydrochloric acid solution with pH=2, so that the collagen mass accounts for 0.65% of the solution mass, and leave it in a refrigerator at 4°C overnight;

[0017] (3) After the collagen sample has fully absorbed water and swelled, transfer it to an ice bath and shear it with a shearing machine at a speed of 3000 rpm until the solution is homogeneous and there is no gel-like precipitate.

[0018] (4) Then, prepare EDC and NHS solutions, put the prepared collagen solution into a beaker, and then add EDC and NHS solutions into the beaker in a mass ratio of collagen:EDC:NHS=12:2:1, stirring while adding to ensure that the reaction is complete.

[0019] (5) Then, add the prepared oxidized dextran solution to the beaker, stirring while adding dropwise to make it evenly mixed. After that, place it on a magnetic stirrer and stir at 300 rpm for 4 hours.

[0020] (6) After stirring, centrifuge the collagen solution at 3000 rpm for 5 minutes to remove air bubbles;

[0021] (7) Then pour the collagen solution into the culture dish at a rate of 45g / dish and use a pipette to remove any remaining air bubbles;

[0022] (8) The culture dish was placed in a biosafety cabinet for air drying to obtain an oxidized dextran modified collagen membrane;

[0023] (9) After air drying, the collagen membrane needs to be repeatedly soaked in deionized water to remove the hydrochloric acid.

[0024] The present invention is further configured such that the concentrations of the EDC and NHS solutions do not exceed 30 mg / ml.

[0025] The present invention is further configured such that the collagen is extracted from bovine Achilles tendon.

[0026] The present invention further provides an oxidized dextran-modified collagen membrane for corneal repair prepared by the above method.

[0027] The present invention further provides the application of the oxidized dextran modified collagen membrane, which is used as a corneal lamellar transplant repair material.

[0028] In summary, the present invention has the following beneficial effects: The present invention selects dextran, which is commonly used as a plasma expander, from biocompatible substances, and performs oxidative grafting on it to give it a certain ability to cross-link with collagen, thereby modifying collagen, improving its mechanical properties, light transmittance and resistance to enzymatic degradation, accelerating cell migration, and thus better applying it to corneal lamellar transplantation repair. Attached Figure Description

[0029] Figure 1 This is a schematic diagram of the synthesis of oxidized dextran in an embodiment of the present invention;

[0030] Figure 2 This is the infrared spectrum of oxidized dextran in an embodiment of the present invention;

[0031] Figure 3 This refers to the actual oxidation degree of ODEX1, ODEX2, and ODEX3 in the embodiments of this invention;

[0032] Figure 4 This refers to the cytotoxicity of oxidized dextran in the embodiments of the present invention;

[0033] Figure 5 This is a diagram illustrating the crosslinking mechanism during the oxidative dextran-modified collagen membrane process in this embodiment of the invention.

[0034] Figure 6 The saturated water content and swelling rate of the collagen membrane and the oxidized dextran-modified collagen membrane in the embodiments of the present invention;

[0035] Figure 7 The optical properties of the collagen membrane and the oxidized dextran-modified collagen membrane in the embodiments of the present invention;

[0036] Figure 8 The mechanical properties of the collagen membrane and the oxidized dextran-modified collagen membrane in the embodiments of the present invention are described.

[0037] Figure 9 These are the degradation curves of the collagen membrane and the oxidized dextran-modified collagen membrane in the embodiments of the present invention;

[0038] Figure 10 These are the CCK8 results of collagen membrane and oxidized dextran-modified collagen membrane in the embodiments of the present invention;

[0039] Figure 11 The results of cell viability and mortality staining of collagen membrane and oxidized dextran modified collagen membrane in the embodiments of the present invention (scale bar: 200 μm).

[0040] Figure 12 This is the cell migration result of collagen membrane and oxidized dextran modified collagen membrane in the embodiments of the present invention (scale bar: 200 μm).

[0041] Figure 13The cell migration rates of the Blank group, Col group, Col-OD1 group, Col-OD3 group, and Col-OD5 group in the embodiments of the present invention are shown below.

[0042] Figure 14 This is a flowchart of the animal experiments in an embodiment of the present invention;

[0043] Figure 15 These are slit-lamp bright-field images of the Blank group, Col group, and Col-OD5 group, which were treated after surgery in different embodiments of the present invention, at 1, 3, 7, 14, 21, and 28 days.

[0044] Figure 16 These are fluorescein sodium staining images of the Blank group, Col group, and Col-OD5 group at 1, 3, 7, 14, 21, and 28 days post-surgery in this embodiment of the invention.

[0045] Figure 17 This is a diagram showing the Col-OD5 and corneal thickness of the normal group 195 days after surgery in an embodiment of the present invention. Detailed Implementation

[0046] The following is in conjunction with the appendix Figure 1-17 The present invention will be described in further detail below.

[0047] Example: A method for preparing an oxidized dextran-modified collagen membrane for corneal repair

[0048] 1.1 Preparation of Oxidized Dextran

[0049] Weigh 5g of dextran and dissolve it in PBS to prepare a dextran aqueous solution. Then, weigh 2.19g, 4.38g, and 6.56g of sodium periodate in the dark and dissolve them in PBS to prepare a sodium periodate solution. Finally, add the dissolved sodium periodate solution dropwise to the dissolved dextran solution (controlling the mass concentration of dextran to be 10%, and the molar ratio of sodium periodate to dextran monomer to be 1:3, 2:3, and 3:3, respectively), stir the reaction in the dark for 3 hours, then add 2ml of ethylene glycol and continue stirring for 1 hour to terminate the reaction. After the reaction is complete, dialyze the resulting solution for 3-4 days and then freeze-dry it to obtain a fluffy, spongy oxidized dextran, which should be sealed and stored in the dark.

[0050] 1.2 Synthesis Principle of Oxidized Glucan

[0051] Oxidized dextran is prepared via sodium periodate. Due to the complexity of the dextran molecule, several different bond-breaking mechanisms exist in this reaction. Studies have shown that when the periodate ion attacks the vicinal diol structure, the amount of C3-C4 bond breaking is 7.5 times that of the C2-C3 bond. Moreover, under mild oxidation conditions, the simultaneous oxidation of C2-C3 and C3-C4 to form formic acid is relatively rare. Furthermore, due to the reactive nature of the aldehyde group, acetals may be formed at high concentrations, interfering with some methods for detecting the aldehyde group. Therefore, hydroxylamine hydrochloride titration is subsequently used to accurately determine the degree of oxidation of oxidized dextran.

[0052] 1.3 Characterization of the prepared oxidized dextran

[0053] (1) Infrared spectrum of oxidized dextran

[0054] Characteristic groups of freeze-dried oxidized dextran were analyzed using Fourier transform infrared spectroscopy (FTIR), with a detection beam range of 4000-400 cm⁻¹. -1 .

[0055] Compared to unmodified pure dextran (Dex), the oxidized product (ODEX1) has a range of ~1740 cm⁻¹. -1 A new characteristic absorption peak appeared. This peak is a typical stretching vibration peak of the C=O bond in an aldehyde group (-CHO). The appearance of this new peak directly proves that sodium periodate successfully oxidized and broke the vicinal diol structure on the dextran ring, converting it into an aldehyde group, thus confirming the successful preparation of oxidized dextran ODEX1. The absorption peaks of ODEX2 and ODEX3 are not obvious, possibly because the high concentration of aldehyde groups underwent an acetal reaction, necessitating further quantitative determination of the degree of oxidation to obtain an accurate value.

[0056] (2) Quantitative determination of the oxidation degree of oxidized dextran

[0057] Prepare a 0.25 mol / L hydroxylamine hydrochloride solution and add methyl orange as an acid-base indicator. Accurately weigh 0.1 g of fully lyophilized oxidized dextran sample (0.1 g dextran for the control group) and add it to 50 ml of hydroxylamine hydrochloride solution. Stir at room temperature for 3 hours to ensure complete dissolution and reaction. After stirring, measure and record the pH value of each solution. Then, titrate the experimental group (oxidized dextran group) with 0.1 mol / L sodium hydroxide solution, measuring the pH value during titration until the pH is the same as the control group, which is the titration endpoint. Record the volume of sodium hydroxide solution consumed at this point, ΔV. The formula for calculating the degree of oxidation η of this batch of oxidized dextran is as follows:

[0058]

[0059] The oxidation degrees of the three oxidized dextrans are denoted as η1, η2, and η3 in ascending order.

[0060] The aldehyde content in oxidized dextran was determined by hydroxylamine hydrochloride titration, and the chemical reaction formula is as follows:

[0061] Dex-(CHO) n + nH2N-OH·HCl→Dex-(CH=N-OH) n + nH2O + nHCl

[0062] HCl + NaOH → NaCl + H2O

[0063] Hydroxylamine hydrochloride reacts with the aldehyde groups in oxidized dextran to form dextran polyoxime (which has almost no effect on titration) and releases an equal amount of HCl as the aldehyde groups. Therefore, the aldehyde content can be calculated by titrating the generated HCl with NaOH and determining the amount of NaOH consumed. With hydroxylamine hydrochloride in excess, the more aldehyde groups there are, the more HCl is generated; therefore, the lower the pH, the redder the corresponding methyl orange color. Using the dextran group as a reference, the aldehyde content of each group can be calculated by obtaining the volume of NaOH consumed when the oxidized dextran group is titrated to the same pH as the dextran group.

[0064] The oxidation degree of the three synthesized oxidized dextrans was determined by NaOH titration, as shown in Table 1. By changing the feed ratio of dextran to sodium periodate, three oxidized dextrans with different oxidation degrees were successfully synthesized.

[0065] Table 1

[0066]

[0067] 1.4 Cellular compatibility of oxidized dextran

[0068] The effect of oxidized dextran at different oxidation levels on the proliferation of RCECs was detected using a CCK-8 assay kit. Cells cultured in wells served as a blank control. The cell seeding density was 1 × 10⁻⁶. 4 Cells / mL were seeded into the plate. On days 1, 3, and 5 after seeding, the cell culture supernatant was aspirated, and the plate was rinsed with PBS to remove excess dead cells and culture medium. The CCK-8 stock solution was diluted with complete culture medium to a concentration of 10%. Under light-protected conditions, 220 μL of CCK-8 working solution was added to each well, ensuring complete immersion of the material. The 48-well plate was then wrapped with aluminum foil and incubated in a cell culture incubator for 3 hours in the dark. After incubation, 100 μL of incubation solution was added to each well of a 96-well plate. Finally, the absorbance (OD) at 450 nm was measured using a microplate reader. The ratio of the net OD value of each group to the net OD value of the plate group is shown below. Figure 4 As shown.

[0069] 2.1 Preparation of Oxyglucan-Modified Collagen Membranes

[0070] Weigh the oxidized dextran prepared in the above steps, dissolve it in deionized water to make a 0.65% aqueous solution, and store it at low temperature and protected from light; weigh the freeze-dried collagen, which is extracted from bovine Achilles tendon and has a molecular weight of about 300,000, cut it into pieces and add it to a hydrochloric acid solution with pH=2, so that the collagen mass accounts for 0.65% of the solution mass, and incubate it overnight at 4°C; after the collagen sample has fully absorbed water and swelled, transfer it to an ice bath and shear it with a shearing machine at a speed of 3000 rpm until the solution is homogeneous and there is no gel-like precipitation.

[0071] Subsequently, EDC and NHS solutions were prepared, with concentrations not exceeding 30 mg / ml. The prepared collagen solution was placed in a beaker, and then EDC and NHS solutions were added dropwise while stirring to ensure complete reaction, according to a mass ratio of collagen (Col):EDC:NHS = 12:2:1. Next, ODEX1 oxidized dextran solution was added to the beaker. The blank group (Col group) did not contain oxidized dextran. In the Col-OD1 group, Col:ODEX1 = 99%:1%; in the Col-OD3 group, Col:ODEX1 = 97%:3%; and in the Col-OD5 group, Col:ODEX1 = 95%:5%. The mixture was stirred dropwise while stirring to ensure homogeneity, and then stirred with a magnetic stirrer at 300 rpm for 4 hours. Figure 4 As shown, oxidized dextran with low oxidation degree has better biocompatibility, so sample ODEX1 was selected for the experiment.

[0072] After stirring, the collagen solution was centrifuged at 3000 rpm for 5 minutes to remove air bubbles. Then, the collagen solution was poured into a culture dish at a rate of 45 g / dish, and any remaining air bubbles were removed with a pipette. The culture dish was then placed in a biosafety cabinet for air drying to obtain an oxidized dextran-modified collagen membrane. After air drying, the obtained collagen membrane needed to be repeatedly soaked in deionized water to remove hydrochloric acid.

[0073] 2.2 Performance Characterization Methods for Oxidized Dextran-Modified Collagen Membranes

[0074] (1) Saturated moisture content

[0075] The saturated water content of the collagen membrane was measured using a gravimetric method. The collagen membrane was cut into circular pieces using a 10mm trephine, and its initial mass M0 and thickness d0 were measured. These pieces were then soaked in physiological saline for 2 hours. Afterward, they were removed, excess solution was wiped off the surface with dry filter paper, and their wet mass M1 and thickness d1 were measured. The saturated water content and swelling ratio of the collagen membrane can then be calculated using the following formulas:

[0076]

[0077]

[0078] The water content and swelling rate of oxidized dextran-modified collagen membranes are directly related to their nutrient permeability and structural stability in the physiological environment. Figure 6 The changes in saturated water content and swelling ratio of collagen membranes modified with different concentrations of oxidized dextran are shown.

[0079] Experimental results showed that both the water content and swelling ratio of the composite membrane exhibited a significant decreasing trend in dependence on ODex concentration. The uncrosslinked pure collagen membrane (Col) exhibited extremely high water absorption, with a water content of approximately 90% and a swelling ratio of about 10 times. However, as the concentration of ODex increased, the water absorption and swelling of the material were significantly suppressed. When the modification concentration reached its maximum (Col-OD5), the water content of the composite membrane decreased to approximately 60%, and the swelling ratio was limited to below 3 times.

[0080] (2) Light transmittance

[0081] Collagen membranes with different ODex contents were placed in physiological saline until they were saturated with water. They were then observed against a clear text background.

[0082] Experimental results show that crosslinking modification significantly improves the light transmittance of the material. The collagen film (Col) without oxidized dextran crosslinking exhibits low light transmittance, with blurred edges on the lettering underneath and noticeable light scattering. Conversely, with increasing oxidized dextran (ODex) crosslinking concentration, the transparency of the composite film achieves a qualitative leap. In particular, the Col-OD3 and Col-OD5 groups exhibit excellent colorless high transparency, with clear outlines of the black lettering at the bottom.

[0083] (3) Mechanical properties

[0084] Collagen membranes with different ODex contents were cut into strips of 15mm × 5mm and stretched at a rate of 0.5N / min using a universal testing machine to obtain stress-strain curves and tensile modulus.

[0085] Experimental results show that crosslinking modification has a significant impact on the mechanical properties of the materials. Uncrosslinked pure collagen membranes (Col) exhibit typical hydrogel characteristics, with low tensile strength (<1.0 MPa) and a high susceptibility to deformation (elongation at break >50%), making it difficult to meet the mechanical performance requirements of corneal materials. After introducing oxidized dextran (ODex), the mechanical strength of the composite membrane shows a significant concentration-dependent nonlinear enhancement.

[0086] With increasing ODex concentration, the initial slope of the stress-strain curve (characterizing Young's modulus / material stiffness) increases significantly. Specifically, the tensile strength at break of the OD5 group experiences an order-of-magnitude jump, reaching approximately 10 MPa.

[0087] (4) Resistance to enzymatic hydrolysis

[0088] To prepare the in vitro enzymatic biodegradation medium for the samples, type I collagenase (China Yuanye Biotechnology Co., Ltd.) was dissolved in physiological saline to achieve an enzyme activity concentration of 5 U / ml (it can be prepared in appropriate batches; unused batches should be stored at -20℃). Collagen membranes with different ODex contents were cut into circular pieces using a 10mm trephine. After saturation in physiological saline, their initial mass M0 was recorded. Each sample was then immersed in 3mL of the enzyme solution and incubated on a shaker at 37℃. At specific time points, the samples were removed from the enzymatic digest, dried, and weighed, denoted as Mt. The prepared collagenase solution was replaced every 12 hours to ensure collagenase activity. The enzymatic hydrolysis resistance curve of the material was obtained by observing the mass changes at different time points.

[0089]

[0090] Experimental results show that cross-linking modification significantly improves the enzymatic resistance of the composite membrane. The uncross-linked pure collagen membrane (Col) has an extremely loose structure and is completely enzymatically degraded within 24 hours, making it completely unsuitable for implantation. The Col-OD1 group, which incorporates a low concentration of cross-linking agent, only managed to extend its degradation time to a mere 48 hours.

[0091] It is worth noting that the materials exhibited excellent long-term stability as the ODex crosslinking density increased further. Although the Col-OD3 group was essentially completely degraded within 168 hours (7 days), there is still a risk of insufficient physical support in the later stages for long-term repair and remodeling of the deep corneal stroma. In contrast, the Col-OD5 group, with the highest crosslinking density, exhibited the best degradation kinetics: after 168 hours, it still retained approximately 40% of the residual stroma network.

[0092] (5) Cell proliferation

[0093] The CCK8 experiment results showed that, during the 1-day, 3-day, and 5-day culture periods, the cells on the surface of all experimental groups (Col, Col-OD1, Col-OD3, Col-OD5) and the blank control group (well plate) maintained a continuous and vigorous proliferation trend, and their OD values ​​showed a healthy gradient increase over time. The Col-OD5 group showed good cell compatibility.

[0094] The growth status of the cells was observed using a Calcein-AM / PI staining kit. First, the kit was removed from a -20°C freezer and centrifuged for 1 min. Then, Calcein-AM and PI solutions were prepared separately with PBS at a ratio of 1:1000 and stored in the dark. After aspirating the supernatant from the cultured cells and rinsing with PBS, 100 μl each of Calcein-AM and PI solutions were added to each well. The wells were then wrapped in aluminum foil and incubated at 37°C in a CO2 incubator for half an hour. The plate was then tilted, and the staining solution was carefully aspirated, followed by rinsing with PBS for 1 min. Finally, the cell status was observed using an inverted fluorescence microscope. Live and dead cells were observed and photographed separately, and the images were then combined to create a complete image of the cell growth status.

[0095] (6) Cell migration

[0096] First, similar to the preparation of materials for cell proliferation, in addition to the above, titanium strips are needed to create scratch patterns. These materials are sterilized with UV light and then soaked in PBS. Next, collagen membranes with different oxidized dextran contents are added to the wells of a plate. The plate is tilted, and a titanium strip is placed on the membrane, secured with a rubber band. 200 μl of complete culture medium is added, and the plate is gently lowered. Cells are digested, centrifuged, and resuspended. The cell suspension is diluted to 12.5 mL and seeded evenly into each well at a rate of 200 μl / well. The culture plate is then placed in a 37°C CO2 incubator, minimizing shaking throughout the process. After 12 hours, when cell adhesion is observed, the titanium strip is immediately removed, and one well is stained with Calcein-AM to observe the cell scratch pattern formation at 0 hours. Subsequently, cell migration is observed periodically using Calcein-AM staining.

[0097] The experimental images clearly demonstrated significant inter-group differences in cell migration efficiency. Observations revealed that the migration rate of the uncrosslinked pure collagen (Col) group was the slowest, with noticeable unclosed gaps remaining even after 60 hours. The introduction of oxidized dextran (ODex) significantly accelerated cell migration, and this promoting effect was positively correlated with the degree of crosslinking. Among all experimental groups, the Col-OD5 group, with the highest crosslinking density and greatest mechanical strength, exhibited exceptionally superior migration-promoting efficacy. At 36 hours, the scratch gaps in the OD5 group had significantly narrowed; by 60 hours, the scratch area in this group was completely covered by an extremely dense cell network, achieving 100% scratch closure, significantly superior to the traditional culture dish control group (Blank) and the low-crosslinking groups.

[0098] 3. Animal experiments using Col-OD5

[0099] First, the animals were anesthetized using a combination of hyaluronic acid and acetaminophen (e.g., Susmin and Sodium 50), and the area around the rabbit's eyes was disinfected with povidone-iodine. A shallow incision was made in the central part of the cornea using a 4 mm corneal trephine, and then the anterior corneal stroma was removed layer by layer using a 15-degree puncture knife to a depth of approximately 180 μm. Subsequently, a pre-polymerized solution of 30% GelMA and 10% ODex was mixed in a 1:1 ratio to obtain a prepolymerized adhesive solution, which was then thoroughly mixed. A small amount of the prepolymerized adhesive solution was then applied to the wound using tweezers. A pre-prepared, thickness-compatible oxidized dextran-modified collagen membrane was then applied to the corneal implantation bed or the corneal injury site. The surface of the material was smoothed with tweezers and then cross-linked and cured using ultraviolet light. An untreated group and a pure collagen membrane group served as controls. Postoperatively, hyaluronic acid eye drops and tobramycin eye drops were administered three times daily, and tobramycin-dexamethasone eye ointment was applied once at night. Results showed that postoperative corneal transparency was well maintained, and no scarring occurred. The material group showed good epithelialization and maintained transparency for a long time. 195 days postoperatively, the cornea remained transparent, and the corneal thickness gradually returned to normal. A transparent stroma had grown over the material.

[0100] This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A method for preparing an oxidized dextran-modified collagen membrane for corneal repair, characterized by: The preparation method includes the following steps: S1. Preparation of oxidized dextran: (1) Weigh out the dextran and dissolve it in PBS to prepare a dextran aqueous solution; (2) Weigh out sodium periodate again in the dark and dissolve it in PBS to prepare a sodium periodate solution; (3) Finally, the above solution was added to a beaker at a ratio of sodium periodate: dextran = 1:

3. The mixture was stirred in the dark for 3 hours, and then a certain amount of ethylene glycol was added and stirred for another 1 hour to terminate the reaction. (4) After the reaction is complete, the resulting solution is dialyzed for 3-4 days and then freeze-dried to obtain fluffy sponge-like oxidized dextran, which is then sealed and stored away from light; S2. Preparation of oxidized dextran-modified collagen membrane: (1) Weigh the oxidized dextran prepared in S1, dissolve it in deionized water to make a 0.65% oxidized dextran aqueous solution, and store it at low temperature and away from light; (2) Weigh the freeze-dried collagen, cut it into pieces and add it to a hydrochloric acid solution with pH=2, so that the collagen mass accounts for 0.65% of the solution mass, and leave it in a refrigerator at 4°C overnight; (3) After the collagen sample has fully absorbed water and swelled, transfer it to an ice bath and shear it with a shearing machine at a speed of 3000 rpm until the solution is homogeneous and there is no gel-like precipitate. (4) Then, prepare EDC and NHS solutions, put the prepared collagen solution into a beaker, and then add EDC and NHS solutions into the beaker in a mass ratio of collagen:EDC:NHS=12:2:1, stirring while adding to ensure that the reaction is complete. (5) Then, add the oxidized dextran aqueous solution to the beaker, stirring while adding dropwise to make it evenly mixed. Then stir with a magnetic stir bar at 300 rpm for 4 hours. (6) After stirring, centrifuge the collagen solution at 3000 rpm for 5 minutes to remove air bubbles; (7) Then pour the collagen solution into the culture dish at a rate of 45g / dish and use a pipette to remove any remaining air bubbles; (8) The culture dish was placed in a biosafety cabinet for air drying to obtain an oxidized dextran modified collagen membrane; (9) After air drying, the collagen membrane needs to be repeatedly soaked in deionized water to remove the hydrochloric acid.

2. The method for preparing oxidized dextran-modified collagen membrane for corneal repair according to claim 1, characterized in that: The concentrations of the EDC and NHS solutions do not exceed 30 mg / ml.

3. The method for preparing oxidized dextran-modified collagen membrane for corneal repair according to claim 1, characterized in that: The collagen was extracted from bovine Achilles tendons.

4. The oxidized dextran-modified collagen membrane for corneal lamellar repair prepared by the method according to any one of claims 1-3.

5. Use of oxidized dextran-modified collagen membranes according to claim 4, characterized in that: Oxidized dextran-modified collagen membranes are used as a repair material for lamellar corneal transplantation.

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