Oxygen-enriched recombinant human type I / V collagen corneal repair gel and preparation method thereof

Abstract

The application discloses an oxygen-enriched recombinant human I / V type collagen corneal repair gel and a preparation method thereof. Methyl methacrylic acid is added into recombinant human I / V type collagen to prepare a methyl methacrylate anhydride recombinant human collagen RHC-I / V-MA solution, the solution is dissolved in an EDC solution for reaction for 2-4 hours to obtain a RHC-I / V-MA-EDC sponge-like solid sample, then the sample is dissolved in a photoinitiator to obtain a mixed solution, an oxygen-containing gas is continuously introduced into the mixed solution by using a micro-nano bubble generator, the flow rate of the oxygen-containing gas is 0.1-2 L / min, and the oxygen-containing gas is introduced for 1-15 minutes to obtain the oxygen-enriched recombinant human I / V type collagen corneal repair gel. The light transmittance of the oxygen-enriched recombinant human I / V type collagen corneal repair gel is extremely high in a spectral range of 400-700 nm, the oxygen-enriched recombinant human I / V type collagen corneal repair gel has good optical stability, is suitable for repairing a corneal transparent layer and provides a material selection with excellent optical performance for corneal repair.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and biomaterials technology, specifically relating to an oxygen-enriched recombinant human type I / V collagen corneal repair gel and its preparation method. Background Technology

[0002] Globally, there are over 400,000 cases of corneal blindness each year. Currently, the main treatments are human donor corneal transplantation or the use of artificial corneas. However, human donor corneas are in severe shortage, and artificial corneas may trigger immune responses, posing a risk of infection. Furthermore, artificial corneas have lower oxygen permeability, leading to suppressed cell metabolism, affecting eye health, and causing various eye problems, including blurred vision and corneal dysfunction. These issues severely limit patients' daily lives and vision recovery. Therefore, effective solutions are urgently needed in the field of corneal repair.

[0003] The main component of the cornea is collagen. Type I collagen not only provides a stable supporting structure for the cornea but also effectively promotes tissue repair and regeneration after corneal damage. Type V collagen plays a crucial role in the exchange of substances and transmission of information between corneal cells and participates in the immune regulation of the cornea. Recombinant human collagen is produced through genetic engineering technology, avoiding the defects of natural collagen. However, traditional hydrogels have high viscosity, making it difficult to form microbubbles during aeration. Large bubbles result in uneven oxygen distribution, failing to fully meet the oxygen requirements for corneal repair and leading to low repair efficiency. Currently, human donor corneal transplant resources are scarce, and artificial corneas have defects such as immune risks and low oxygen permeability. The field of corneal repair urgently needs innovative breakthroughs. Summary of the Invention

[0004] To address the issues of immune risks and low oxygen permeability associated with traditional corneal repair materials, this invention provides a method for preparing an oxygen-enriched recombinant human collagen corneal repair gel, which utilizes a micro / nano bubble generator to create an oxygen-enriched environment. Furthermore, this invention provides an oxygen-enriched recombinant human type I / V collagen corneal repair gel, which uses recombinant human type I / V collagen as its core ingredient. This gel achieves its oxygen-enriched environment through precise control of the material ratio and the use of a micro / nano bubble generator.

[0005] The present invention also provides the application of the above-mentioned oxygen-enriched recombinant human type I / V collagen corneal repair gel.

[0006] The technical solution adopted in this invention is as follows:

[0007] A method for preparing an oxygen-enriched recombinant human collagen corneal repair gel includes the following steps:

[0008] 1) Methacrylic acid was added to the recombinant human collagen solution and stirred to prepare a methacrylic anhydride-modified recombinant human collagen RHC-MA solution. The supernatant was collected by centrifugation and freeze-dried to obtain a sponge-like solid sample of RHC-MA.

[0009] 2) Dissolve the freeze-dried RHC-MA sponge-like solid sample in 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution and react for 2-4 h. Then filter the solution, dialyze, and freeze-dry to obtain RHC-MA-EDC sponge-like solid.

[0010] 3) Weigh out the RHC-MA-EDC sponge-like solid and dissolve it in the photoinitiator. Mix well to obtain a 5-50% RHC-MA-EDC solution, which is the precursor solution for the oxygen-rich corneal repair gel.

[0011] 4) Using a micro-nano bubble generator, oxygen-containing gas is continuously introduced into the mixed solution of step 3), with a gas flow rate of 0.1 L / min-2 L / min and a treatment time of 1 min-15 min, to obtain oxygen-enriched recombinant human collagen corneal repair gel.

[0012] In step 1), the concentration of the recombinant human collagen (RHC) solution is 20-60 mg / mL.

[0013] The recombinant human collagen solution is obtained by adding recombinant human collagen to PBS solution and stirring to dissolve it.

[0014] Step 1) The recombinant human collagen is at least one of recombinant human type I, type II, type III, and type V collagen, and may also include collagen, gelatin, polypeptides, and recombinant collagen extracted from animals.

[0015] In step 1), the amount of methacrylic acid solution added is 3%-20% (v / v) of the volume of the recombinant human collagen solution.

[0016] In step 2), the concentration of the EDC solution is 0.1 M-1 M, preferably 0.5 M.

[0017] In step 2), the freeze-dried RHC-MA sponge-like solid sample is dissolved in EDC solution for 2-4 hours, preferably 3 hours.

[0018] Recombinant human collagen is any one of recombinant human type I, II, III, and V collagen, and sponge-like solids RHC-I-MA-EDC, RHC-II-MA-EDC, RHC-III-MA-EDC, and RHC-V-MA-EDC are prepared respectively.

[0019] In step 4), the oxygen concentration in the oxygen-containing gas is 20-100%, more preferably 23-100%; the remaining gas is N2 or an inert gas such as Ar; preferably, the oxygen-containing gas contains 30% O2 and 70% N2; the oxygen concentration is preferably 30% to ensure that oxygen is slowly released in the gel, prolonging the oxygen-enriched time and balancing the repair effect and cell safety.

[0020] The oxygen-enriched recombinant human collagen corneal repair gel prepared by the above method.

[0021] The recombinant human collagen is preferably recombinant human type I collagen and recombinant human type V collagen, which can be used to prepare an oxygen-enriched recombinant human type I / V collagen corneal repair gel. The preparation method includes the following steps:

[0022] 1) Methacrylic acid was added to the recombinant human type V collagen solution and stirred to prepare a methacrylic anhydride recombinant human type V collagen RHC-V-MA solution. The supernatant was collected by centrifugation and lyophilized to obtain a sponge-like solid sample of RHC-V-MA. The sponge-like solid sample of RHC-I-MA was prepared by the same method.

[0023] 2) The freeze-dried RHC-V-MA sponge-like solid sample was dissolved in 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution and reacted for 2-4 h. The solution was then filtered, dialyzed, and freeze-dried to obtain the RHC-V-MA-EDC sponge-like solid sample. The RHC-I-MA-EDC sponge-like solid sample was prepared by the same method.

[0024] 3) Weigh out the sponge-like solid samples of RHC-I-MA-EDC and RHC-V-MA-EDC, dissolve them in the photoinitiator, and mix them evenly to obtain a mixed solution;

[0025] 4) Using a micro-nano bubble generator, oxygen-containing gas is continuously introduced into the mixed solution of step 3), with a gas flow rate of 0.1 L / min-2 L / min and an introduction time of 1 min-15 min, to obtain oxygen-enriched recombinant human type I / V collagen corneal repair gel.

[0026] Preferably, the recombinant human type I / V collagen solution in step 1) is prepared by dissolving recombinant human type I / V collagen powder in PBS buffer to obtain a recombinant human type I / V collagen solution with a concentration of 20-60 mg / mL; more preferably, it is a recombinant human type I / V collagen solution with a concentration of 50 mg / mL (i.e., 5% w / v).

[0027] In step 1), the methacrylic acid solution is 3%-20% (v / v) of the volume of the recombinant human type I / V collagen solution, preferably 6% (v / v).

[0028] The photoinitiator mentioned in step 3) can be irgacure 2959 or lithium phenyl (2,4,6-trimethylbenzoyl)phosphate (LAP), preferably LAP.

[0029] The concentration of the photoinitiator mentioned in step 3) is 0.1%-2% (w / v), preferably 0.1%-1% (w / v), and more preferably 0.2% (w / v).

[0030] In step 3), different proportions of RHC-I-MA-EDC and RHC-V-MA-EDC sponge solids are dissolved in the photoinitiator, with the ratio range being (m)RHC-I-MA-EDC:(m)RHC-V-MA-EDC=10:1-1:10, preferably (m)RHC-I-MA-EDC:(m)RHC-V-MA-EDC=8:2.

[0031] In step 3), the RHC-V-MA-EDC and RHC-I-MA-EDC are dissolved in the photoinitiator at a concentration of 5%-50%; more preferably, the concentration is 10-30%, and more preferably, the concentration is 30% (w / v).

[0032] Preferably, RHC-I-MA-EDC and RHC-V-MA-EDC are dissolved in 10 mL of 0.2% (w / v) photoinitiator at a ratio of 8:2, i.e., 2.4 g of RHC-I-MA-EDC and 0.6 g of RHC-V-MA-EDC. The preferred concentration is 30% (w / v).

[0033] In step 4), the flow rate of the continuously introduced gas is 0.1 L / min to 2 L / min, preferably 1-2 L / min, and the continuous gas introduction treatment time is preferably 1 min-15 min, more preferably 3 min.

[0034] The oxygen-enriched recombinant human type I / V collagen corneal repair gel prepared by the aforementioned method.

[0035] The oxygen-enriched recombinant human type I / V collagen corneal repair gel is used in the preparation of corneal replacement products, soft or rigid contact lenses, corneal damage repair materials, or materials that promote the proliferation of corneal-related cells.

[0036] This invention utilizes recombinant human type I / V collagen to synthesize corneal repair gel. A systematic study was conducted on different ratios of type I / V materials to test their effects on the gel's physicochemical properties, biocompatibility, and corneal repair efficacy. By adjusting the content of recombinant human type I / V collagen, an artificial corneal material with similar biomechanical properties and tissue compatibility was prepared to better simulate the structure and function of the natural cornea. Then, a micro / nano bubble generator was used to provide an oxygen-rich environment, further enhancing the corneal repair effect.

[0037] The recombinant human collagen corneal repair gel prepared in this invention exhibits high fluidity and can form atomized microbubbles during ventilation, significantly increasing the contact area between oxygen and the gel. This continuous oxygen supply creates a favorable aerobic environment for corneal cell repair and regeneration, meeting the oxygen requirements of cell metabolism and promoting cell proliferation and differentiation. Simultaneously, due to the gel's stable and moderately sized three-dimensional network structure, the stable sustained-release mechanism prevents rapid oxygen loss, extending the gel's effective action time and demonstrating significant application potential in the field of corneal repair.

[0038] Oxygen concentration is a key factor affecting the corneal repair effect. Too low an oxygen concentration cannot meet the vigorous metabolic needs of corneal cells, thus hindering the healing process of corneal tissue; too high an oxygen concentration may trigger cytotoxic reactions and damage corneal cells. The performance of recombinant human collagen corneal repair gel is closely related to ventilation time and flux: if the ventilation time is too short, the gel will not be sufficiently oxygenated and will be unable to meet the oxygen requirements of corneal repair; if the time is too long, it will easily cause excessive oxidation of gel components. In terms of flux, low flux leads to poor oxygen diffusion and uneven distribution, while high flux will damage the microstructure and rheological properties of the gel. In this invention, the ventilation time is preferably 1 min-15 min, more preferably 3 min, and the oxygen concentration is preferably 30%.

[0039] This invention explores the optimal performance of recombinant human collagen corneal repair gel. It requires comprehensive consideration of the effects of factors such as oxygen concentration, recombinant human collagen concentration, I / V ratio, optimal ventilation time and flux on corneal repair, in order to precisely control the relevant parameters so that the mechanical properties of the gel match the needs of corneal repair, achieve a balance between biocompatibility and mechanical support, and provide better material support for corneal repair treatment.

[0040] Firstly, this invention selects recombinant human type I and type V collagen as core raw materials, leveraging the supportive and repairing role of type I and the cell-regulating role of type V to solve the problems of traditional materials. The resulting recombinant human type I / V collagen composite gel has excellent optical properties, biocompatibility, and mechanical properties.

[0041] Secondly, the present invention provides a method for preparing oxygen-enriched recombinant human type I / V collagen composite gel, wherein the micro-nano bubble aeration head of a micro-nano bubble generator is inserted into a mixed solution of RHC-V-MA-EDC and RHC-I-MA-EDC, and oxygen is continuously introduced to generate a large number of micro and nano-sized oxygen bubbles.

[0042] Thirdly, this invention provides an oxygen-enriched corneal repair gel that slowly releases oxygen. The recombinant human type I / V collagen has high fluidity and forms atomized microbubbles during ventilation, increasing the contact area between oxygen and the gel. The stable three-dimensional network structure enables slow oxygen release, preventing rapid dissipation, fully meeting the metabolic needs of corneal cells, accelerating repair and delaying tissue degeneration, improving the application of oxygen supply to corneal cells, reducing corneal cell damage, and reducing degradation caused by low oxygen permeability. At the same time, the oxygen-enriched material can reduce foreign body sensation and improve user comfort.

[0043] In the fourth aspect, this invention provides the application of an oxygen-enriched corneal repair gel. Oxygen supply promotes corneal health and metabolism, helps improve corneal healing ability, accelerates cell regeneration and wound repair, thereby shortening healing time and reducing complications caused by low corneal oxygen permeability, such as vascularization and inflammation. Simultaneously, it provides multiple molding methods, including static molding, centrifugal casting, molding, and 3D printing, to meet different design and application needs.

[0044] Beneficial effects:

[0045] The oxygen-enriched recombinant human type I / V collagen gel prepared in this invention exhibits biocompatibility similar to that of natural human cornea, forming a good adhesion interface on the surface of corneal epithelial cells and significantly reducing the occurrence of immune rejection. In corneal epithelial cell survival tests, the material showed a high survival rate, indicating that it can be stably survived by corneal cells for a long time, providing a good foundation for corneal repair or replacement materials.

[0046] Oxygen-enriched recombinant human type I / V collagen gel has extremely high light transmittance in the 400-700 nm spectral range and good optical stability. This characteristic makes it suitable for the repair or replacement of the corneal lamina, providing a material option with excellent optical performance for corneal repair.

[0047] Oxygen-enriched recombinant human type I / V collagen gel exhibits good mechanical stability in compression tests and can withstand normal stress in corneal tissue. Its excellent mechanical properties make it suitable for corneal repair or replacement materials.

[0048] Oxygen-enriched recombinant human type I / V collagen gel can promote the proliferation and differentiation of corneal epithelial cells and support the corneal repair process. In addition, the oxygen-enriched cross-linked gel can mimic the tissue structure and function of the natural cornea, providing an ideal material model for corneal defect repair. Attached Figure Description

[0049] Figure 1 Experimental flowchart;

[0050] Figure 2 Physical properties of RHC-I-MA lyophilized products, including: (a) RHC-I-MA lyophilized products; (b) injectability; (c) corneal repair gel; and (d) light transmittance.

[0051] Figure 3 The corneal prosthesis formed by the curing of oxygen-enriched corneal repair gel includes: (a) a simulated corneal trephine; (b) an injected and stained corneal repair gel; (c) the corneal repair gel curing after ultraviolet light irradiation; (d) the corneal repair gel being picked up with tweezers; and (e) the stretchability of the corneal repair gel.

[0052] Figure 4 Comparison of bubble distribution after 10 s, 1 min, and 2 min of ventilation using RHCMA and GELMA (bubbles are represented by dashed boxes);

[0053] Figure 5 After ventilation with GELMA (left) and RHCMA (right) and resting for 1 min and 3 min respectively;

[0054] Figure 6 Distribution of RHCMA and GELMA bubbles after ventilation and standing for 1 min and 3 min (scale bar: 20 μm);

[0055] Figure 7 GELMA and RHCMA bubble size distribution diagrams after 1 min of ventilation;

[0056] Figure 8 Stress-strain curves of the sample under different compression rates in Example 3;

[0057] Figure 9 Maximum stress and Young's modulus of the sample in Example 3;

[0058] Figure 10 Absorbance of RHCMA gel samples at different concentrations;

[0059] Figure 11 Oxygen content at different ventilation times;

[0060] Figure 12 Oxygen release curve after 10 minutes of ventilation;

[0061] Figure 13 Gel condition of 0.3% (w / v), 4% (w / v), and 5% (w / v) oxygen-enriched corneal repair gel precursor solutions after UV irradiation;

[0062] Figure 14 CCK-8 assay results of cell viability after treatment with different concentrations of RHCMA extract;

[0063] Figure 15 Cell fluorescence staining results after culturing with different concentrations of RHCMA extract (scale bar: 20 μm);

[0064] Figure 16 CCK-8 assay results of cell viability after treatment with RHCMA extract at different ventilation rates;

[0065] Figure 17 Cell fluorescence staining results after culturing in RHCMA extract at different ventilation rates (scale bar: 20 μm);

[0066] Figure 18 RHCMA seeded at different ventilation rates, cell fluorescence staining results (scale bar: 20 μm). Detailed Implementation

[0067] The present invention will be further described below with reference to specific embodiments and accompanying drawings.

[0068] Recombinant human type I collagen lyophilized powder was purchased from Jiangsu Jiangshan Juyuan Biotechnology Co., Ltd.

[0069] The recombinant human type V collagen described in this invention can be prepared by genetically engineered bacteria, such as patent CN118702807A; it can also be purchased commercially. In the following examples, the recombinant human type V collagen lyophilized powder was purchased from Jiangsu Jiangshan Juyuan Biotechnology Co., Ltd.

[0070] The reagents used in the following examples are all conventional reagents in the art and can be purchased commercially.

[0071] Example 1: Preparation method of oxygen-enriched recombinant human type I / V collagen corneal repair gel

[0072] 1) Weigh 500 mg of recombinant human type V collagen and add it to 10 mL of PBS solution. Stir for 10 minutes to completely dissolve it, and prepare a 5% (w / v) aqueous solution of recombinant human type V collagen (50 mg / mL). Add 6% (w / v) methacrylic acid solution to the aqueous solution of recombinant human type V collagen and stir to obtain RHC-V-MA solution. Collect the supernatant by centrifugation, dilute, filter, dialyze, and freeze-dry to obtain RHC-V-MA sponge-like solid sample. Prepare RHC-I-MA sponge-like solid sample in the same way.

[0073] 2) The freeze-dried RHC-V-MA sponge-like solid sample was dissolved in 0.5 M 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution and reacted for 4 h. The solution was then filtered, dialyzed, and freeze-dried to obtain the RHC-V-MA-EDC sponge-like solid sample. The RHC-I-MA-EDC sponge-like solid sample was prepared by the same method.

[0074] 3) Weigh out a sponge-like solid with a ratio of (m)RHC-V-MA-EDC:(m)RHC-I-MA-EDC = 2:8 and dissolve it in a 0.2% (w / v) photoinitiator solution (prepare a 0.2% (w / v) photoinitiator solution by dissolving 20 mg of LAP powder in 10 mL of PBS solution). Mix thoroughly.

[0075] 4) Insert the micro-nano bubble aeration head of the micro-nano bubble generator into the mixed solution and continuously introduce oxygen-containing gas (30% O2 and 70% N2, percentages are by volume). The gas flow rate is 2 L / min, and the treatment time is 3 min to obtain the corneal repair gel precursor solution, which is then stored.

[0076] A circular corneal defect model was prepared using a corneal trephine. A stained corneal repair gel precursor solution was injected into the defect area to facilitate observation of its distribution. After irradiation with ultraviolet light, the gel solidified to form a corneal repair. The experimental procedure is as follows: Figure 1 As shown, the scientific validity, operability, and suitability for clinical application of the preparation method are verified. Figure 2 The results demonstrated the RHC-I-MA lyophilized product and its injectability, showcasing the intermediate product's process stability and ease of clinical use, as well as the corneal repair gel's morphological uniformity and optical properties. Figure 3 A simulated corneal trephine was used to inject stained corneal repair gel. After UV irradiation, the corneal repair gel solidified. The corneal repair gel was then picked up with tweezers, and its stretchability was verified. At the same time, the clinical suitability, mechanical stability and operational tolerance of the product were verified.

[0077] Example 2: Comparison of aeration bubble characteristics between traditional GELMA and recombinant human type I / V collagen precursor solution

[0078] GELMA: Dissolve 40 mg of LAP powder in 20 mL of PBS solution to prepare a 0.2% (w / v) photoinitiator solution (LAP PBS solution). Add 1.2 g of GELMA to 20 mL of LAP PBS to obtain a 6% (w / v) GELMA solution. Store in the dark.

[0079] Recombinant human type I / V collagen precursor solution: 6% (v / v) methacrylic acid was added to a recombinant human type I / V collagen solution (50 mg / mL), and the mixture was stirred to prepare a methacrylic anhydride-modified recombinant human collagen RHC-I / V-MA solution. The supernatant was collected by centrifugation and lyophilized to obtain a sponge-like solid sample of RHC-I / V-MA. The lyophilized sponge-like solid sample of RHC-I / V-MA was dissolved in 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution and reacted for 2-4 h. The solution was then filtered, dialyzed, and lyophilized to obtain a sponge-like solid of RHC-I / V-MA-EDC. 20 mg of LAP powder was dissolved in 10 mL of PBS solution to prepare a 0.2% (w / v) photoinitiator solution. Weigh 0.8g of RHC-I-MA-EDC and 0.2g of RHC-V-MA-EDC sponge solids and dissolve them in 10mL of photoinitiator. Mix them evenly to obtain a 10% (w / v) RHC-I / V-MA-EDC solution, which is the precursor solution for the oxygen-enriched corneal repair gel.

[0080] Take 5 mL of solution in the same test tube, purge at a flow rate of 1 L / min for 5 min, insert a micro / nano bubble aerator, and record the bubble characteristics. The results are as follows: Figure 4 As shown, after 10 seconds, 1 minute, and 2 minutes of aeration, RHCMA produced finer bubbles, while GELMA produced larger and less uniform bubbles. After 3 minutes of aeration and settling, as shown... Figure 5 As shown, RHCMA defoams faster while GELMA defoams slowly.

[0081] After ventilation was stopped, images were taken, with three non-overlapping fields of view randomly selected from each group. Microscopic images were used to measure bubble size (≥100 particles per group) in ImageJ, and the range, average particle size, and distribution density were statistically analyzed; results are as follows. Figure 6 The figures show the bubble distribution of GELMA and RHCMA after 1 min and 3 min of ventilation, respectively. The bubble size of GELMA bubbles is significantly larger than that of RHCMA bubbles. A bubble size distribution diagram was created after 1 min of ventilation, as shown below. Figure 7 As shown, the RHCMA peaks are concentrated in the 5-10 μm range, with a smaller standard deviation (3.11) and a more concentrated particle size distribution. The GELMA peaks are concentrated around 15 μm, with a larger standard deviation (5.94) and a more dispersed particle size distribution. The average particle size of GELMA (16.74 μm) is much larger than that of RHCMA (6.67 μm).

[0082] Example 3: Effects of different concentrations of RHC-MA on the mechanical stability, transmittance, and rheological properties of oxygen-enriched corneal repair gel

[0083] 6% (v / v) methacrylic acid was added to a recombinant human type I / V collagen solution (50 mg / mL), and the mixture was stirred to prepare a methacrylic anhydride-modified recombinant human collagen RHC-I / V-MA solution. The supernatant was collected by centrifugation and lyophilized to obtain a sponge-like solid sample of RHC-I / V-MA. The lyophilized sponge-like solid sample of RHC-I / V-MA was dissolved in 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution and reacted for 2-4 h. The solution was then filtered, dialyzed, and lyophilized to obtain a sponge-like solid of RHC-I / V-MA-EDC. 20 mg of LAP powder was dissolved in 10 mL of PBS solution to prepare a 0.2% (w / v) photoinitiator solution. Weigh 0.8g of RHC-I-MA-EDC and 0.2g of RHC-V-MA-EDC sponge solids and dissolve them in 10mL of photoinitiator. Mix well to obtain a 10% (w / v) RHC-I / V-MA-EDC solution. Similarly, weigh 1.6g of RHC-I-MA-EDC and 0.4g of RHC-V-MA-EDC sponge solids and dissolve them in 10mL of photoinitiator. Mix well to obtain a 20% (w / v) RHC-I / V-MA-EDC solution. Weigh 2.4g of RHC-I-MA-EDC and 0.6g of RHC-V-MA-EDC sponge solids and dissolve them in 10mL of photoinitiator. Mix well to obtain a 30% (w / v) RHC-I / V-MA-EDC solution. Thus, 10% (w / v), 20% (w / v), and 30% (w / v) oxygen-enriched corneal repair gel precursor solutions are prepared.

[0084] The micro-nano bubble aeration head of the micro-nano bubble generator was inserted into the oxygen-enriched corneal repair gel precursor solution to continuously introduce gas at a flow rate of 1 L / min for 10 min. The oxygen-enriched corneal repair gel precursor solution was then injected into a centrifuge tube and irradiated with ultraviolet light for 1 minute until it was completely gelled.

[0085] The compressive modulus was tested using a universal testing machine with a strain rate of 1 mm / min. The stress-strain curves for each sample at different compression rates were recorded, and the compressive modulus was calculated based on the stress-strain curves. Figure 8 It is known that 10% (w / v) and 20% (w / v) RHC-MA have low stiffness and a gradual stress increase, while 30% (w / v) RHC-MA has high stiffness and a large maximum stress. Therefore, the higher the proportion of RHC-MA, the higher the material's stiffness and strength, but the lower its plastic deformation capacity. Figure 9 It can be seen that the maximum stress increases significantly, the Young's modulus increases exponentially, and the increase in the RHC-MA ratio has statistical reliability in enhancing mechanical properties.

[0086] To test transmittance, the UV-irradiated gel was cut into uniform slices with a thickness of 0.1 mm and a diameter of 10 mm using a sterile blade (ensuring a smooth, bubble-free surface; three parallel samples were prepared for each group). These slices were then immersed in PBS at 37°C for 24 h to simulate a physiological environment. The gel slices were fixed in quartz cuvettes, and baseline correction was performed on blank cuvettes (containing only PBS at 37°C) to eliminate solvent interference. The absorbance of RHC-MA gel samples at different concentrations was measured sequentially, and the transmittance curves in the range of 400-700 nm were recorded (transmittance = (sample absorbance / reference absorbance) × 100%). The results are as follows: Figure 10 As shown, the RHC-MA ratio mainly affects the transmittance at short wavelengths (350–450 nm), while the transmittance of each group is similar and excellent at long wavelengths (450–750 nm).

[0087] Example 4: Effect of different ventilation times on the oxygen content of oxygen-enriched corneal repair gel

[0088] Similar to Example 3, a 10% (w / v) oxygen-enriched corneal repair gel precursor solution was prepared. The micro-nano bubble aeration head of a micro-nano bubble generator was inserted into the solution to continuously introduce gas (here, gas refers to a mixture of oxygen and nitrogen at 30% and 70%). The gas flow rate was 0.1 L / min, and the treatment times were 0 s, 10 s, 30 s, 60 s, 180 s, 240 s, 300 s, 420 s, and 600 s.

[0089] Oxygen content was tested using a dissolved oxygen meter under different ventilation times, with each test performed three times. Results are as follows: Figure 11 As shown, the oxygen content gradually increases with increasing ventilation time; when the ventilation time reaches 240 seconds, the oxygen content remains relatively stable and no longer increases significantly, indicating that oxygen saturation has been achieved. The optimal ventilation time is 240 seconds (4 minutes).

[0090] The ventilation time was set to 10 minutes. Oxygen levels were measured at 10 min, 1 h, 3 h, 5 h, 8 h, 19 h, and 24 h after ventilation. Figure 12 After 10 minutes of oxygenation, the oxygen content of the system was significantly higher than the initial level without oxygenation (4.22 mg / L). However, as time continued (from 1 h to 24 h), the oxygen content continued to decrease. By 24 hours, the oxygen content had basically returned to the initial state without oxygenation (4.22 mg / L). This indicates that the system can maintain a relatively large amount of oxygen for a longer period of time, and oxygen was slowly released within 24 hours.

[0091] Example 5: Biocompatibility Verification Experiment of Oxygen-Enriched Corneal Repair Gel at Different Concentrations

[0092] Similar to Example 3, 3% (w / v), 4% (w / v), and 5% (w / v) oxygen-enriched corneal repair gel precursor solutions were prepared. Their gelation was observed under ultraviolet light to determine the minimum concentration required for gel formation. Figure 13 As shown, (a) has a concentration of 3% (w / v) and is in a liquid state, not gelled; (b) has a concentration of 4% (w / v) and is partially gelled; and (c) has a concentration of 5% (w / v) and is completely gelled. Therefore, 5% (w / v) is the critical gelation value for the precursor solution of oxygen-enriched corneal repair gel.

[0093] Similar to Example 3, 10% (w / v), 20% (w / v), and 30% (w / v) oxygen-enriched corneal repair gel precursor solutions were prepared respectively. The three different concentrations of oxygen-enriched corneal repair gel precursor solutions were filtered and sterilized through a 0.4 μm sterilization membrane. Aeration treatment: The micro-nano bubble aeration head of the micro-nano bubble generator was inserted into the mixed solution of each concentration group and continuously introduced at a flow rate of 1 L / min for 10 min.

[0094] After aeration, the solutions of each concentration group were injected into centrifuge tubes and irradiated with ultraviolet light for 1 minute until they were completely gelled.

[0095] Biocompatibility validation was performed by preparing hydrogel extracts according to international biocompatibility standards ISO 10993-5 and ISO 10993-12. The appropriate culture media were used at 3 cm⁻¹. 2 The hydrogel sample was immersed at an extraction ratio of / mL and incubated at 37℃ for 24 h, after which the extract was collected. Human corneal epithelial cells (HCECs) adherent to Dulbecco's Modified Eagle Medium (DMEM) were digested, resuspended, and seeded at a density of 3000 cells / well in 96-well plates. 100 μL of DMEM medium was added to each well, and the plates were incubated for 12 h to allow complete cell adhesion. The medium was then replaced with 100 μL of sample extract, and the plates were incubated for another 24 h. Normal medium was used as a negative control. After 24 h of incubation, 10 μL of CCK-8 was added to each well, and the plates were incubated at 37℃ for 2 h. After incubation, absorbance was measured at 450 nm using a microplate reader. Figure 14 As shown, the cell viability values ​​at different concentrations of RHCMA were similar, indicating good biocompatibility.

[0096] Live and dead cells were stained using Calcein-AM / PI. The morphology of HCEC cells cultured for specific durations in different RHCMA extraction media was observed. A staining working solution was prepared by mixing 5 mL Assay Buffer (1×), 5 μL Calcein-AM solution (2 mM), and 15 μL PI solution (1.5 mM). After culturing cells in the extraction media for 24 h and 48 h, the cells were washed three times with Assay Buffer to thoroughly remove residual esterase activity. Then, 100 μL of PBS was added to each well, followed by 50 μL of the staining working solution, and the cells were incubated at 37°C for 15 min. After incubation, the cell morphology after fluorescence staining was observed using a fluorescence microscope. Figure 15 As shown, the cells are morphologically intact and evenly distributed, further verifying that the RHCMA extract has little interference with the cell growth microenvironment and good biocompatibility.

[0097] Example 6: Experimental Study on the Supporting Effect of Oxygen-Enriched Corneal Repair Gels with Different Airflow Rates on the Proliferation of Human Corneal Epithelial Cells

[0098] Same as in Example 3, prepare a 10% (w / v) oxygen-enriched corneal repair gel precursor solution; filter it through a 0.4 μm sterilization membrane for sterilization.

[0099] Aeration treatment: Insert the micro-nano bubble aeration head of the micro-nano bubble generator into the mixed solutions of each concentration group and aerate for 10 min. Aeration flow rates: 0 L / min, 0.5 L / min, 1 L / min, 2 L / min.

[0100] After aeration, the solutions of each concentration group were injected into centrifuge tubes and irradiated with ultraviolet light for 1 minute until they were completely gelled.

[0101] Hydrogel extracts were prepared according to the international biocompatibility standards ISO 10993-5 and ISO 10993-12. The appropriate culture media were used at 3 cm⁻¹. 2 The hydrogel sample was immersed in an extraction ratio of / mL and placed in a cell culture incubator at 37 ℃ for 24 h before the extract was collected.

[0102] Human corneal epithelial cells (HCECs) adherent to DMEM medium were digested, resuspended, and seeded at a density of 3000 cells / well in 96-well plates, with three replicates per group. 100 μL of DMEM medium was added to each well, and the plates were incubated for 12 h to allow complete cell adhesion. The medium was then replaced with 100 μL of sample extract, and the plates were incubated for another 24 h. Normal medium was used as a negative control. After 24 h of incubation, 10 μL of CCK-8 was added to each well, and the plates were incubated at 37°C for 2 h. After incubation, absorbance was measured at 450 nm using a microplate reader. Figure 16 As shown, cell viability remained consistently high at 70%–80% with stable data, indicating low cytotoxicity and good biocompatibility.

[0103] Live and dead cells were stained using Calcein-AM / PI. The morphology of HCEC cells cultured for specific durations in oxygen-enriched corneal repair gel extract (a liquid obtained by extracting specific components from solid raw materials through dissolution and diffusion during the extraction process) under different ventilation flow rates was observed. A staining working solution was prepared by mixing 5 mL Assay Buffer (1×), 5 μL Calcein-AM solution (2 mM), and 15 μL PI solution (1.5 mM). After culturing cells in the extract for 24 h and 48 h, the cells were washed three times with Assay Buffer to thoroughly remove residual esterase activity. Then, 100 μL of PBS was added to each well, followed by 50 μL of the staining working solution, and the cells were incubated at 37°C for 15 min. Cell morphology after fluorescence staining was observed using a fluorescence microscope after incubation. Figure 17 As shown, under different ventilation flow rates, the morphology was intact and the distribution was uniform, with no obvious apoptosis or necrosis, further verifying that it has good biocompatibility.

[0104] Add 80 μL of RHCMA at different oxygen flow rates (0 L / min, 0.5 L / min, 1 L / min, 2 L / min) to 96-well plates. After UV curing for 40 seconds, collect HCEC cells in logarithmic growth phase that have adhered to the plate, digest them with trypsin, centrifuge, and resuspend them in DMEM medium. Adjust the cell density to 3000 cells / well. Add 80 μL of cell suspension to each well of the gel, with 3 replicates per group. Incubate in a cell culture incubator for 24 h. Mix 5 mL of Assay Buffer (1×), 5 μL of Calcein-AM solution (2 mM), and 15 μL of PI solution (1.5 mM) thoroughly and set aside. Wash three times with AssayBuffer, add 100 μL of PBS to each well to maintain the cell environment, then add 50 μL of staining working solution; incubate at 37°C in the dark for 15 min; observe under a fluorescence microscope: Calcein-AM labeled live cells (green fluorescence), PI labeled dead cells (red fluorescence), and record cell morphology by photograph. Figure 18 As shown, at an aeration rate of 0-2 L / min, HCECs survived well on the gel surface with no significant accumulation of dead cells, demonstrating good biocompatibility.

Claims

1. A method for preparing an oxygen-enriched recombinant human type I / V collagen corneal repair gel, characterized in that, Includes the following steps: 1) Methacrylic acid was added to the recombinant human type V collagen solution and stirred to prepare a methacrylic anhydride recombinant human type V collagen RHC-V-MA solution. The supernatant was collected by centrifugation and freeze-dried to obtain a sponge-like solid sample of RHC-V-MA. The sponge-like solid sample of RHC-I-MA was prepared by the same method. 2) The freeze-dried RHC-V-MA sponge-like solid sample was dissolved in 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution and reacted for 2-4 h. The solution was then filtered, dialyzed, and freeze-dried to obtain the RHC-V-MA-EDC sponge-like solid sample. The RHC-I-MA-EDC sponge-like solid sample was prepared by the same method. 3) Weigh out the RHC-I-MA-EDC sponge-like solid sample and the RHC-V-MA-EDC sponge-like solid sample, dissolve them in the photoinitiator, and mix them evenly to obtain a mixed solution; 4) Using a micro-nano bubble generator, oxygen-containing gas is continuously introduced into the mixed solution of step 3), with a gas flow rate of 0.1-2 L / min and an introduction time of 1-15 min, to obtain oxygen-enriched recombinant human type I / V collagen corneal repair gel.

2. The method for preparing an oxygen-enriched recombinant human type I / V collagen corneal repair gel according to claim 1, characterized in that, In step 1), the concentration of the recombinant human type V collagen solution is 20-60 mg / mL.

3. The method for preparing an oxygen-enriched recombinant human type I / V collagen corneal repair gel according to claim 1, characterized in that, In step 1), the amount of methacrylic acid added is 3%-20% of the volume of the recombinant human type V collagen solution.

4. The method for preparing an oxygen-enriched recombinant human type I / V collagen corneal repair gel according to claim 1, characterized in that, The photoinitiator mentioned in step 3) is lithium phenyl (2,4,6-trimethylbenzoyl) phosphate or irgacure 2959.

5. The method for preparing an oxygen-enriched recombinant human type I / V collagen corneal repair gel according to claim 1, characterized in that, In step 3), the RHC-V-MA-EDC sponge solid sample and the RHC-I-MA-EDC sponge solid sample are mixed at a mass ratio of 1:10-10:

1.

6. The method for preparing an oxygen-enriched recombinant human type I / V collagen corneal repair gel according to claim 1, characterized in that, The oxygen content in the oxygen-containing gas described in step 4) is 23%-100%, and the percentage is a volume percentage.

7. The method for preparing an oxygen-enriched recombinant human type I / V collagen corneal repair gel according to claim 1, characterized in that, In step 4), the gas flow rate is 1-2 L / min, and the gas introduction time is 1-15 min.

8. The oxygen-enriched recombinant human type I / V collagen corneal repair gel prepared by any one of the preparation methods of claims 1 to 7.

9. The use of the oxygen-enriched recombinant human type I / V collagen corneal repair gel according to claim 8 in the preparation of corneal replacement products, soft contact lenses or rigid contact lenses.

10. The use of the oxygen-enriched recombinant human type I / V collagen corneal repair gel according to claim 8 in the preparation of corneal damage repair materials or materials that promote the proliferation of corneal-related cells.

Images