A biomimetic layered corneal repair gel and methods of making and using same

Abstract

The application provides a kind of biomimetic layered corneal repair gel and its preparation method and application, the biomimetic layered gel includes upper layer gel and lower layer gel;Upper layer gel and lower layer gel are combined at volume ratio 1:9 to 5:5;Upper layer gel and lower layer gel each have three-dimensional crosslinked network;Upper layer gel and lower layer gel are crosslinked with each other to form an integrated structure between each other.Upper layer gel biomimics corneal basement membrane structure, can promote cell regeneration and differentiation;Lower layer gel biomimics corneal stroma, has rigidity, anti-degradation and other characteristics, to provide support for upper layer gel.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to a biomimetic layered corneal repair gel, its preparation method, and its application. Background Technology

[0002] In the field of corneal repair, liquid hydrogel materials have become a research hotspot due to their advantages such as high cost-effectiveness, injectability, and seamless implantation. For example, patent document TW202229419A discloses a corneal occlusion and tissue adhesion material based on methacrylamide gelatin (GelMA) and its mixtures; patent document WO2025097846A1 proposes an in-situ corneal filler composed of methacrylic acid succinic acid diesterized starch and methacrylamide gelatin; and patent document CN118105542A introduces a photocurable biomaterial constructed by preparing collagen fibers through electrospinning and introducing photopolymerizable groups.

[0003] However, existing hydrogel materials still fall short of meeting the multi-dimensional performance requirements for corneal defect repair. For example, collagen-based methacrylamide materials (such as GelMA) possess excellent biocompatibility and can promote epithelial cell regeneration and wound healing, but they are easily degraded rapidly by collagenases in vivo, making them unable to fill corneal defects and provide stable mechanical support in the long term. Conversely, synthetic materials such as methacrylamide polyethylene glycol (PEGDA) can effectively resist enzymatic degradation and provide long-term structural support and mechanical stability, but they lack cell recognition sites, making it difficult to induce wound tissue regeneration and healing. Simply mixing the two can achieve some performance benefits in the short term, but this leads to functional dilution, and the mismatch in degradation rates between the two phases causes internal structural instability and premature disintegration of the material.

[0004] Therefore, developing corneal repair materials with different properties to address different corneal injuries and clinical needs remains a challenge in this field. Summary of the Invention

[0005] To overcome the shortcomings of the prior art, in a first aspect, the present invention provides a biomimetic layered gel, comprising an upper gel and a lower gel;

[0006] The upper gel includes methacrylamide gelatin, collagen, laminin (LAM), photoinitiator, and coupling agent.

[0007] The lower gel includes polyethylene glycol diacrylate or chondroitin sulfate with methacrylamide, as well as gelatin, photoinitiator, and coupling agent;

[0008] The upper and lower gels each have a three-dimensional cross-linked network;

[0009] The upper and lower gels are combined in a volume ratio of 1:9 to 5:5;

[0010] The upper and lower gels form an integrated structure. This integrated structure refers to molecular-level cross-linking and interpenetration, with no physical interfaces, no component phase separation, and no delamination within the system, forming a continuous, uniform, and stable three-dimensional network.

[0011] In a second aspect, the present invention provides a method for preparing a biomimetic layered gel, comprising the following steps:

[0012] a) Prepare the upper layer composite crosslinking solution and the lower layer composite crosslinking solution;

[0013] The upper composite cross-linking solution comprises 10%-20% (w / v) methacrylamide gelatin, 5%-20% (w / v) collagen, 0.8 mg / mL-1.2 mg / mL laminin, 0.2%-0.3% photoinitiator, and 0.05%-0.2% coupling agent; the lower composite cross-linking solution comprises 5%-20% (v / v) polyethylene glycol diacrylate or 5%-20% (w / v) methacrylamide chondroitin sulfate, 5%-10% (w / v) gelatin, 0.2%-0.3% photoinitiator, and 0.1%-0.5% coupling agent.

[0014] b) After the upper composite crosslinking solution and the lower composite crosslinking solution react separately, an upper solid gel and a lower solid gel with a three-dimensional crosslinking network are obtained;

[0015] c) The upper solid gel and the lower solid gel are subjected to hydrothermal treatment to obtain fluid upper gel and lower gel;

[0016] d) Combine the upper and lower gels in a volume ratio of 1:9 to 5:5 to bring the two gels into contact and form a covalent bond, thus obtaining a biomimetic layered gel.

[0017] Preferably, the collagen material includes gelatin, type I / III collagen, recombinant human collagen, etc.

[0018] Preferably, the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP), sodium phenyl-2,4,6-trimethylbenzoylphosphonate (NAP), or a combination thereof;

[0019] Preferably, the coupling agent is a combination of carbodiimide and an N-hydroxy compound, wherein the carbodiimide is selected from one or more of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, and 1-cyclohexyl-2-morpholinoethylcarbodiimide; and the N-hydroxy compound is selected from one or more of N-hydroxysuccinimide and sulfonyl-N-hydroxysuccinimide.

[0020] Preferably, the coupling agent is a triazineonium salt coupling agent, including 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinonium salt;

[0021] Preferably, the coupling agent is a ureonium / phosphoniumium salt coupling agent, selected from one or more of O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate, benzotriazol-1-yl-oxytripyrrolylphosphine hexafluorophosphate, and (benzotriazol-1-yloxy)tri(dimethylamino)phosphonium hexafluorophosphate.

[0022] Preferably, the molecular weight of polyethylene glycol diacrylate is 500-10000.

[0023] Preferably, in step b), the reaction conditions are standing at 10-34°C; in step c), the hydrothermal treatment is a water bath at 40-150°C.

[0024] Preferably, the viscosity of the upper gel and the lower gel is 2000-10,000 mPa·s.

[0025] In a third aspect, the present invention provides a biomimetic layered gel prepared according to the above-described method for preparing a biomimetic layered gel.

[0026] In the above-mentioned biomimetic layered gel, the cross-linking density of the lower gel is higher than that of the upper gel; the upper gel has a porous and loose network structure (biomimetic corneal basement membrane structure), while the lower gel has a fibrous network structure (biomimetic natural corneal stroma structure); the network pore size of the lower gel is smaller than that of the upper gel.

[0027] Preferably, the biomimetic layered gel has an overall transmittance of ≥80%, a refractive index of 1.33-1.40, and a tensile strength of ≥20 kPa.

[0028] In a fourth aspect, the present invention provides the use of the above-mentioned biomimetic layered gel in the preparation of medical devices for repairing corneal damage; or in the preparation of cell carriers or drug carriers.

[0029] Preferably, the corneal injury includes ocular surface injury, corneal epithelial injury, corneal stromal injury, corneal ulcer, corneal perforation, corneal laceration, etc.

[0030] Beneficial effects

[0031] 1. Excellent biomimetic performance: This invention replicates the natural layered structure and mechanical differences of the cornea. Through component design, it achieves tissue regeneration promotion in the upper layer and strong degradation resistance in the lower layer, precisely matching the physiological needs of the cornea.

[0032] 2. Resolving the core contradiction: By using a layered design, the functions of collagen-based materials and synthetic materials are complemented, avoiding the performance imbalance caused by simple mixing. This ensures the long-term mechanical support and degradation stability of the materials, while also promoting epithelial regeneration and wound healing, thus achieving long-term repair.

[0033] 3. Strong clinical applicability: The material is injectable and photocurable, allowing for seamless implantation. It is easy to operate, reduces patient trauma and infection risks, and shortens the recovery period. At the same time, the preparation process is controllable, and the component concentration and reaction conditions can be adjusted according to the defect, adapting to the repair needs of different types of corneal defects.

[0034] 4. Excellent structural stability: Through CMC / NHS crosslinking and layered perfusion technology, the upper and lower gel layers form a tightly integrated structure with no obvious cracks, ensuring the mechanical continuity of the material and avoiding postoperative delamination. Attached Figure Description

[0035] Figure 1 The structural characterization of the biomimetic layered hydrogel includes: A. Visualization of the layered hydrogel: The layered structure is visually presented in a canned state through red staining of the upper layer and green staining of the lower layer (the lower right corner shows a picture of the actual gel). After extrusion, the gel maintains a viscoelastic fluid state with layering, preserving the layered morphology. There is no diffusion or miscibility between the layers, and it exhibits good resistance to tear / aqueous water erosion. B. Hematoxylin-eosin staining results of the cured layered hydrogel, scale bar: 200 μm. After curing, the two gel layers form a seamless, continuous, and complete integrated interface without obvious gaps or structural interruptions. C. Scanning electron microscope image of the cured layered hydrogel: The white transition zone marks the self-fusion interface between the upper and lower layers. The interface is tightly bonded without separation. The upper layer is a porous and loose network, while the lower layer is a relatively dense network structure, scale bar: 50 μm. D. Transmission electron microscope image of the upper layer of the layered hydrogel, scale bar: 300 nm. E. Transmission electron microscope image of the lower layer of the layered hydrogel, scale bar: 300 nm.

[0036] Figure 2 Results of cell compatibility and healing-promoting effects of the upper and lower gel layers, including: A. Live and dead cell staining results of human corneal epithelial and stromal cells cultured on the gel surface for 48 hours, with green fluorescence representing live cells and red fluorescence representing dead cells, scale bar: 100 μm; B. Representative cell scratch experiment photographs showing the promotion of corneal epithelial cell migration by the upper and lower gel layers, scale bar: 200 μm; C. Quantitative results of relative cell viability detected by CCK-8 assay; D. Quantitative statistical results of scratch healing area of ​​the upper and lower gel layers.

[0037] Figure 3The results of the intraocular pressure tolerance test of the biomimetic layered gel include: A. Schematic diagram of the artificial anterior chamber model simulating intraocular pressure test: The corneal substitute material was fixed on the artificial anterior chamber device, and the pressure was monitored by injecting air with a syringe and a pressure meter to evaluate the pressure resistance of the material; B. Morphological changes of materials of different embodiments under 80 mmHg pressure; C. Quantitative results of burst pressure of materials of different embodiments: The burst pressure of Example 1 was significantly higher than that of Example 4 (***p<0.001, **p<0.01).

[0038] Figure 4 The in vivo effects of different filling materials on repairing lamellar defects of rabbit cornea, including: A. Slit-lamp microscopy observation results at 7 and 28 days postoperatively; B. Optical coherence tomography (OCT) images; C. Hematoxylin-eosin staining results, scale bar: 200 μm. Detailed Implementation

[0039] The cornea, as a vital refractive tissue on the ocular surface, possesses a sophisticated layered structure, composed sequentially of the epithelium, Bowman's layer, stroma, Descemet's membrane, and endothelium. Furthermore, the anterior and posterior halves of the corneal stroma exhibit significant structural and functional differences. This layered characteristic is the core foundation for the cornea's optical function and mechanical stability. Its significance lies in the synergistic adaptation of the differences in structure and performance among the layers to achieve the three core functions of optical transparency, mechanical stability, and tissue compatibility, thus providing effective protection for intraocular tissues and ensuring normal visual function.

[0040] This invention utilizes the advantages of both collagen-based and synthetic materials to prepare a biomimetic gel with a layered structure, mimicking the natural layered structure and performance differences of the cornea to achieve long-term repair and tissue regeneration of corneal defects.

[0041] The biomimetic layered gel provided by this invention comprises an upper gel and a lower gel. The upper gel mimics the structure of the corneal basement membrane and can promote cell regeneration and differentiation. The lower gel mimics the corneal stroma and has characteristics such as rigidity and resistance to degradation, providing support for the upper gel. The two are integrated into a single structure through layered infusion and curing.

[0042] The upper gel mainly consists of methacrylamide gelatin (GelMA), collagen (Col), laminin (LAM), photoinitiator, and coupling agent.

[0043] Preferably, the concentration of methacrylamide gelatin is 10%-20% (w / v), the concentration of collagen is 5%-20% (w / v), the concentration of laminin is 0.8 mg / mL-1.2 mg / mL, the concentration of photoinitiator is 0.2%-0.3%, and the concentration of coupling agent is 0.05%-0.2%.

[0044] Preferably, the collagen material includes, but is not limited to, gelatin, type I / III collagen, recombinant human collagen, etc.

[0045] Preferably, the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP), sodium phenyl-2,4,6-trimethylbenzoylphosphonate (NAP), or a combination thereof.

[0046] Preferably, the coupling agent includes, but is not limited to, any one or a combination of the following compounds: (a) a combination system of carbodiimide and N-hydroxy compound, wherein the carbodiimide is selected from 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N,N'-dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC) and / or 1-cyclohexyl-2-morpholinoethylcarbodiimide (CMC); the N-hydroxy compound is selected from N-hydroxysuccinimide (NHS), sulfonyl-N-hydroxysuccinimide (sulfon-NHS, Sulfo-NHS) and / or hydroxybenzotriazole (HOBt); or (b) a single-component triazineonium salt activator, such as 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinonium salt; or (c) Ureonium / phosphonium salt coupling agents, such as O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU), benzotriazol-1-yl-oxytripyrrolylphosphonium hexafluorophosphate (PyBOP), and / or (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP).

[0047] The lower gel mainly consists of polyethylene glycol diacrylate (PEGDA) or chondroitin sulfate methacrylamide, gelatin (Cel), photoinitiator, and coupling agent.

[0048] Preferably, the concentration of polyethylene glycol diacrylate is 5%-20% (v / v) or chondroitin sulfate with methacrylamide is 5%-20% (w / v), the concentration of gelatin is 5%-10% (w / v), the concentration of photoinitiator is 0.2%-0.3%, and the concentration of coupling agent is 0.1%-0.5%.

[0049] Preferably, the molecular weight of polyethylene glycol diacrylate is 500-10000; more preferably, the molecular weight is 500-5000, such as 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500.

[0050] Preferably, the gelatin is biomedical grade gelatin, derived from bovine bone / pigskin, with a gel strength ≥250 Bloom.

[0051] Preferably, the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP), sodium phenyl-2,4,6-trimethylbenzoylphosphonate (NAP), or a combination thereof.

[0052] Preferably, the coupling agent includes, but is not limited to, any one or a combination of the following compounds: (a) a combination system of carbodiimide and N-hydroxy compound, wherein the carbodiimide is selected from 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N,N'-dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC) and / or 1-cyclohexyl-2-morpholinoethylcarbodiimide (CMC); the N-hydroxy compound is selected from N-hydroxysuccinimide (NHS), sulfonyl-N-hydroxysuccinimide (sulfon-NHS, Sulfo-NHS) and / or hydroxybenzotriazole (HOBt); or (b) a single-component triazineonium salt activator, such as 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinonium salt; or (c) Ureonium / phosphonium salt coupling agents, such as O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU), benzotriazol-1-yl-oxytripyrrolylphosphonium hexafluorophosphate (PyBOP), and / or (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP).

[0053] The steps for preparing the above-mentioned biomimetic layered gel include (wherein, the preparation steps for the upper and lower gels are basically the same, except for the different grouping and concentration):

[0054] a) Prepare the upper layer composite crosslinking solution and the lower layer composite crosslinking solution;

[0055] The upper composite crosslinking solution comprises 10%-20% (w / v) methacrylamide gelatin, 5%-20% (w / v) collagen, 0.8 mg / mL-1.2 mg / mL laminin, 0.2%-0.3% photoinitiator, and 0.05%-0.2% coupling agent; the lower composite crosslinking solution comprises 5%-20% (v / v) polyethylene glycol diacrylate or 5%-20% (w / v) methacrylamide chondroitin sulfate, 5%-10% (w / v) gelatin, 0.2%-0.3% photoinitiator, and 0.1%-0.5% coupling agent.

[0056] b) After the upper composite crosslinking solution and the lower composite crosslinking solution react separately, an upper solid gel and a lower solid gel with a three-dimensional crosslinking network are obtained;

[0057] c) The upper solid gel and the lower solid gel are subjected to hydrothermal treatment to obtain a viscoelastic fluid-like upper gel and lower gel.

[0058] d) Combine the upper and lower gels in a volume ratio of 1:9 to 5:5 to make the two gels come into contact and form a connection, thus obtaining a biomimetic layered gel.

[0059] Preferably, the collagen material includes, but is not limited to, gelatin, type I / III collagen, recombinant human collagen, etc.

[0060] Preferably, the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP), sodium phenyl-2,4,6-trimethylbenzoylphosphonate (NAP), or a combination thereof.

[0061] Preferably, the coupling agent includes, but is not limited to, any one or a combination of the following compounds: (a) a combination system of carbodiimide and N-hydroxy compound, wherein the carbodiimide is selected from 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N,N'-dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC) and / or 1-cyclohexyl-2-morpholinoethylcarbodiimide (CMC); the N-hydroxy compound is selected from N-hydroxysuccinimide (NHS), sulfonyl-N-hydroxysuccinimide (sulfon-NHS, Sulfo-NHS) and / or hydroxybenzotriazole (HOBt); or (b) a single-component triazineonium salt activator, such as 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinonium salt; or (c) Ureonium / phosphonium salt coupling agents, such as O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU), benzotriazol-1-yl-oxytripyrrolylphosphonium hexafluorophosphate (PyBOP), and / or (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP).

[0062] Preferably, the molecular weight of polyethylene glycol diacrylate is 500-10000; more preferably, the molecular weight is 500-5000, such as 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500.

[0063] Preferably, the gelatin is biomedical grade gelatin, derived from bovine bone / pigskin, with a gel strength ≥250 Bloom.

[0064] Step b) constructs a continuous, complete, and water-insoluble three-dimensional cross-linked network at the molecular level. This network is the structural basis for the material's permanent "waterproof," "solubility-resistant," and "dilution-resistant" properties, which are maintained by the adhesives used in subsequent processing and applications.

[0065] Step c) Inducing controlled, partial hydrolytic breakage of amide bonds under high-temperature (40-150°C) hydrothermal conditions systematically reduces the crosslinking density and imparts deformability to the gel without disrupting the overall network continuity. By precisely controlling temperature and time, the macroscopic state of the gel can be repeatedly and accurately controlled from a solid elastomer to a fluid with appropriate viscoelasticity, facilitating subsequent minimally invasive delivery and application.

[0066] In step d), the upper gel and the lower gel spontaneously fuse at the contact interface to form an integrated layered gel with a stable structure and continuous interface.

[0067] Preferably, the volume ratio of the upper and lower gel layers is 1:9 to 5:5 to balance the biomimetic design and function. A higher upper layer ratio (e.g., 5:5) is more conducive to epithelial regeneration and wound healing, but the overall degradation rate may be accelerated; a higher lower layer ratio (e.g., 1:9) enhances mechanical support and anti-degradation properties. Therefore, the ratio can be adaptively adjusted according to the depth, location, and clinical repair goals of the defect.

[0068] This invention does not limit the contact area, contact time, or contact environment; those skilled in the art can adjust these parameters according to actual conditions. The interface between the two gel layers should be made as close as possible to avoid air bubbles or gaps. At room temperature or physiological temperature, interface self-healing can usually be completed within minutes. It is recommended to operate under sterile conditions, with room temperature to 37°C and suitable humidity, to avoid drying or excessive cooling that could affect the mobility of the polymer chain segments.

[0069] The biomimetic layered gel prepared in this invention mimics the natural layered structure and performance differences of the cornea, achieving long-term repair and regeneration of corneal defects. The upper gel mimics the basement membrane structure, promoting cell regeneration and differentiation; the lower gel mimics the corneal stroma, possessing rigidity and degradation resistance, providing support for the upper gel. During use, after photocuring, the material rapidly forms an integrated network structure, achieving immediate and strong adhesion to wet tissue, thereby completing the sealing and repair functions. It exhibits transparency, erosion resistance, and biocompatibility, making it particularly suitable for ophthalmological fields with extremely high requirements for intraoperative visual field and postoperative optical quality.

[0070] The layered gel provided by this invention includes, but is not limited to, the following properties:

[0071] 1. Optical performance

[0072] Light transmittance: ≥80% in the visible light band (400-700 nm); refractive index: 1.33-1.40, matching the refractive index of aqueous humor and corneal stroma.

[0073] 2. Rheological properties before curing

[0074] Viscosity: 2000-10000 mPa·s (25℃), can be injected through a 22G-27G sterile needle.

[0075] 3. Mechanical properties after curing

[0076] Tensile strength: Overall tensile strength of the integrated gel ≥20 kPa; Shear strength: ≥30 kPa; Intraocular pressure tolerance: Bursting pressure ≥100 mmHg.

[0077] 4. Degradation performance

[0078] Upper gel: enzymatically degraded, with a degradation cycle of 3-4 months, matching the corneal epithelial regeneration cycle, and gradually degraded after regeneration is completed;

[0079] The lower gel layer is hydrolyzed and degraded, with a degradation cycle of 6-18 months to a year. It provides long-term mechanical support for corneal stroma regeneration and avoids tissue collapse after repair.

[0080] Degradation products: amino acids, ethylene glycol, and water; no residue remains, and can be metabolized and excreted by the body.

[0081] 5. Cell-guided tissue regeneration

[0082] Upper gel: Promotes human corneal epithelial cell (HCEC) adhesion rate ≥95%, increases proliferation rate by 20%-30%, and can form continuous stratified epithelium within 7 days;

[0083] Lower gel: non-cytotoxic (cell viability ≥96%).

[0084] In some preferred embodiments, the preparation method of the biomimetic layered gel includes the following steps:

[0085] (a) Preparation of the upper gel

[0086] 1.1 Prepare an aqueous solution of upper-layer photoinitiator with a concentration of 0.2-0.3% (w / v).

[0087] The photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP), sodium phenyl-2,4,6-trimethylbenzoylphosphonate (NAP), or a combination thereof. This invention does not limit the solvent, but water or phosphate buffered saline (PBS) is preferred.

[0088] 1.2 Preparation of the upper composite solution, for example, dissolving methacrylamide gelatin (GelMA), collagen (Col), and laminin (LAM) in an aqueous photoinitiator solution to obtain a uniform and transparent GelMA-Col-LAM composite solution (GC solution for short).

[0089] The collagen material is gelatin or recombinant human collagen.

[0090] The laminin is a laminin extracted from animal tissue or expressed through genetic engineering recombination, such as laminin derived from mouse sarcoma or recombinant human laminin.

[0091] 1.3. Prepare an upper layer coupling agent solution with a mass-volume fraction of 10-30%.

[0092] The present invention does not limit the solvent, but water or phosphate buffer (PBS) is preferred.

[0093] The coupling agent includes, but is not limited to, compounds selected from any one of the following or combinations thereof: (a) a combination system of carbodiimide and N-hydroxy compound, wherein the carbodiimide is selected from 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N,N'-dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC) and / or 1-cyclohexyl-2-morpholinoethylcarbodiimide (CMC); the N-hydroxy compound is selected from N-hydroxysuccinimide (NHS), sulfonyl-N-hydroxysuccinimide (sulfon-NHS, Sulfo-NHS) and / or hydroxybenzotriazole (HOBt); (b) a single-component triazineonium salt activator, such as 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinonium salt (DMTMM); or (c) Ureonium / phosphonium salt coupling agents, such as O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU), benzotriazol-1-yl-oxytripyrrolylphosphonium hexafluorophosphate (PyBOP), and / or (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP).

[0094] This invention does not limit the specific operations of solvent preparation, mixing, shaking, sterilization, filtration, and storage; those skilled in the art can choose according to the actual situation. The embodiments described in this invention are some embodiments of this invention, but not all embodiments.

[0095] The present invention does not restrict the order of preparation of each solution in steps 1.1-1.3, and those skilled in the art can adjust it according to the actual situation.

[0096] 1.4 Mix the GC solution and the upper coupling agent solution to obtain the upper composite crosslinking solution.

[0097] Preferably, the main components of the upper composite crosslinking solution include: 0.2-0.5% photoinitiator, 10%-20% (w / v) GelMA, 5%-20% (w / v) Col, 0.8 mg / mL-1.2 mg / mL LAM, and 0.3-0.5% (w / v) coupling agent.

[0098] Preferably, the mixing involves slowly adding a layer of coupling agent solution dropwise to the GC solution under continuous rapid mechanical stirring or vortex conditions. After the addition is complete, mixing continues (e.g., for 1-2 minutes) to ensure uniform dispersion.

[0099] Preferably, the process further includes a degassing step: removing air bubbles from the upper composite crosslinking solution to ensure the uniformity and optical transparency of the subsequently formed gel. For example, the upper composite crosslinking solution is transferred to a sterile centrifuge tube, placed in a preheated centrifuge, and centrifuged at 8000-12000 rpm for 1-2 minutes.

[0100] This invention does not limit the specific operations of mixing and degassing; those skilled in the art can choose according to the actual situation. The embodiments described in this invention are some, but not all, of the embodiments.

[0101] 1.5. The upper composite cross-linking solution undergoes a cross-linking reaction to obtain an upper solid gel with a three-dimensional network structure.

[0102] Preferably, the reaction conditions are standing at 10–34°C.

[0103] The present invention does not limit the reaction time, for example, from 10 minutes to 2 hours.

[0104] This step has a significant impact on the dilution resistance and stability of the final gel, enabling it to more effectively resist the immediate flushing and dilution by bodily fluids such as tears, aqueous humor, and blood.

[0105] 1.6. Heat the upper solid gel until a viscoelastic fluid-like upper gel is obtained.

[0106] Preferably, the heating method is a water bath at 40-80℃; the heating time is 5-20 hours.

[0107] In this step, the viscosity of the upper gel is adjusted to a suitable level according to actual needs through real-time monitoring. For example, the dynamic viscosity of the final product is precisely controlled within the range of 2000-10,000 mPa·s. Once the viscosity reaches the predetermined range, heating is immediately stopped, and the gel is cooled (e.g., at room temperature or 4°C) to obtain the upper gel.

[0108] (II) Preparation of the lower gel layer

[0109] 2.1 Prepare a lower layer photoinitiator aqueous solution with a concentration of 0.2-0.3% (w / v).

[0110] The photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP), sodium phenyl-2,4,6-trimethylbenzoylphosphonate (NAP), or a combination thereof. This invention does not limit the solvent, but water or phosphate buffered saline (PBS) is preferred.

[0111] 2.2 Preparation of the lower layer composite solution, for example, adding gelatin and PEGDA or gelatin and chondroitin sulfate methacrylamide to the photoinitiator aqueous solution to form a PEGDA-gelatin composite solution or a chondroitin sulfate methacrylamide-gelatin composite solution.

[0112] The gelatin is a biomedical grade gelatin, derived from bovine bone / pigskin, with a gel strength ≥250 Bloom.

[0113] 2.3 Prepare a lower layer coupling agent solution with a mass-volume fraction of 10-30%.

[0114] The present invention does not limit the solvent, but water or phosphate buffer (PBS) is preferred.

[0115] The coupling agent includes, but is not limited to, compounds selected from any one of the following or combinations thereof: (a) a combination system of carbodiimide and N-hydroxy compound, wherein the carbodiimide is selected from 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), 1-cyclohexyl-2-morpholinoethylcarbodiimide (CMC); and the N-hydroxy compound is selected from N-hydroxysuccinimide (NHS), sulfonyl-N-hydroxysuccinimide (sulfon-NHS, Sulfo-NHS), and / or hydroxybenzotriazole (HOBt); (b) Single-component triazine salt activators, such as 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium salt (CMC / NHS); or (c) ureonium / phosphonium salt coupling agents, such as O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU) and benzotriazol-1-yl-oxytripyrrolidinephosphide hexafluorophosphate (PyBOP).

[0116] This invention does not limit the specific operations of solvent preparation, mixing, shaking, sterilization, filtration, and storage; those skilled in the art can choose according to the actual situation. The embodiments described in this invention are some embodiments of this invention, but not all embodiments.

[0117] The present invention does not restrict the order of preparation of each solution in steps 2.1-2.3, and those skilled in the art can adjust it according to the actual situation.

[0118] 2.4 Mix the PG solution and the lower coupling agent solution to obtain the lower composite crosslinking solution.

[0119] Preferably, the main components of the lower composite crosslinking solution include: 0.2-0.5% photoinitiator, 5%-20% (v / v) PEGDA or chondroitin sulfate with methacrylamide, 5%-10% (w / v) gelatin (Cel), and 0.05%-0.1% (w / v) coupling agent;

[0120] Preferably, the mixing involves slowly adding the lower layer coupling agent solution dropwise to the PG solution under continuous rapid mechanical stirring or vortex conditions. After the addition is complete, mixing continues (e.g., for 1-2 minutes) to ensure uniform dispersion.

[0121] Preferably, the process further includes a degassing step: removing air bubbles from the lower composite crosslinking solution to ensure the uniformity and optical transparency of the subsequently formed gel. For example, the lower composite crosslinking solution is transferred to a sterile centrifuge tube, placed in a preheated centrifuge, and centrifuged at 8000-12000 rpm for 1-2 minutes.

[0122] This invention does not limit the specific operations of mixing and degassing; those skilled in the art can choose according to the actual situation. The embodiments described in this invention are some, but not all, of the embodiments.

[0123] 2.5. The lower layer of composite cross-linking solution undergoes a cross-linking reaction to obtain a lower layer solid gel with a three-dimensional network structure.

[0124] Preferably, the reaction conditions are standing at 10-34°C.

[0125] The present invention does not limit the reaction time, for example, from 10 minutes to 2 hours.

[0126] 2.6. Heat the lower solid gel until a viscoelastic fluid-like lower gel is obtained.

[0127] Preferably, the heating method is a water bath at 60-110℃; the heating time is 5-20 hours.

[0128] In this step, the lower gel is adjusted to a suitable viscosity as needed through real-time monitoring. For example, the dynamic viscosity of the final product is precisely controlled within the range of 2000-10,000 mPa·s. Once the viscosity reaches the predetermined range, heating is immediately stopped, and the gel is cooled (e.g., at room temperature or 4°C) to obtain the upper gel.

[0129] (III) Combining the upper and lower gel layers

[0130] By combining the upper and lower gels in a volume ratio of 1:9 to 5:5, the two gels come into contact and form a stable interface, thus obtaining a biomimetic layered gel.

[0131] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0132] While similar or equivalent steps, substances or materials, and reaction conditions may be used in the implementation of this invention, preferred steps, substances or materials, and reaction conditions are described herein.

[0133] When a range of values ​​is described herein, unless otherwise stated, the range is intended to include its endpoints and all integers and fractions within that range, and all values ​​within that range are capable of achieving the effects of the present invention.

[0134] Unless otherwise stated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by one of ordinary skill in the art or the field of application of such terms.

[0135] As used herein, the singular form of a word includes the plural, and vice versa. Therefore, “a,” “an,” and “the” generally include the plural form of the corresponding term. As used herein, “an embodiment” or “embodiment” refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the invention. The phrase “in one embodiment” appearing in different places throughout this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0136] As used herein, “resistant,” “none,” “low,” or “reduced” means a state of resistance, reduction, or disappearance, or a reduction in a detectable or observable quantity. In some embodiments, one of the assessment tools described herein is used to measure this reduction or disappearance. In some embodiments, resistance or absence, or reduction or decrease, indicates a difference.

[0137] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available or conventionally prepared products.

[0138] Some of the raw materials include: methacrylamide gelatin (GelMA, 90% substitution), type I collagen (Col, derived from bovine Achilles tendon), laminin (LAM, derived from mouse sarcoma), N-acrylomorpholine (NAP, 98% purity), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (CMC / NHS, 99% purity), polyethylene glycol diacrylate (PEGDA700, number average molecular weight 700), gelatin (Cel, biomedical grade), L-phenylalanine (LAP, 99% purity), and phosphate-buffered saline (PBS, pH=7.4), all of which are commercially available biomedical grade raw materials.

[0139] Preparation equipment: sterile biosafety cabinet, constant temperature water bath, high-speed refrigerated centrifuge, visible light curing instrument (wavelength 450nm), 0.22μm sterile filter membrane, sterile syringe (1mL, 5mL), corneal repair and reshaping device.

[0140] Example 1

[0141] (a) Preparation of the upper gel

[0142] The upper gel was prepared with methacrylamide gelatin (GelMA), collagen (Col), and laminin (LAM) as core components, using phenyl (2,4,6-trimethylbenzoyl)lithium phosphate (LAP) aqueous solution as solvent and 1-cyclohexyl-2-morpholinoethyl carbodiimide (CMC) / N-hydroxysuccinimide (NHS) as coupling agent. The final concentrations of each component were: 20% (w / v) GelMA, 10% (w / v) collagen, 1 mg / mL laminin, 0.3% LAP, and 0.2% (w / v) CMC / NHS. The specific preparation steps are as follows:

[0143] 1. Solvent preparation: Prepare the upper LAP aqueous solution as the base solvent for dissolving the core components.

[0144] 2. Preparation of the base solution: Add GelMA, Col and LAM powders sequentially to the upper LAP solution prepared in step 1. Place the mixture in a water bath at 40±2℃ and magnetically stir at 100-200 rpm for 1-2 hours until all components are completely dissolved to obtain a homogeneous and transparent GelMA-Col-LAM composite solution (GC solution for short).

[0145] 3. Preparation of the upper coupling agent solution: Prepare the CMC / NHS solution fresh for use. The specific method is as follows: add CMC / NHS powder to sterile phosphate buffer (PBS, pH 7.2-7.4) and vortex for 30-60 seconds until completely dissolved.

[0146] 4. Preparation of the upper layer composite crosslinking solution: Take the GC solution prepared in step 2 into a sterile centrifuge tube. Quickly add the CMC / NHS solution prepared in step 3 using a micropipette, and immediately use the pipette tip to blow up and down 10-15 times to ensure rapid mixing.

[0147] 5. Degassing treatment: Place the mixed solution from step 4 in a centrifuge preheated to 37-40°C and centrifuge at 8000-12000 rpm for 1-2 minutes to remove air bubbles introduced during the mixing process.

[0148] 6. Dispensing and Sealing: Carefully transfer the degassed, clear solution into a 2-3 mL sterile syringe. Install a 0.22 μm sterile filter and injection needle, and slowly inject the solution through the filter into a 1 mL sterile syringe. Immediately cap the syringe with the rubber stopper to prevent contamination.

[0149] 7. Gelation treatment: Place the syringe containing the solution at room temperature (20-25℃) for 10-20 minutes to allow for initial cross-linking and the formation of a solid gel. Then, place it vertically in a constant temperature water bath at 50-60℃ for hot hydrolysis reaction for 8-10 hours. After the reaction is complete, store the resulting upper gel in a refrigerator at 4℃ for later use.

[0150] (II) Preparation of the lower gel layer

[0151] The lower gel was prepared using polyethylene glycol diacrylate (PEGDA, molecular weight 700) and gelatin (Cel) as core structural components, lithium phenyl (2,4,6-trimethylbenzoyl)phosphate (LAP) as a photoinitiator, and CMC / NHS as an auxiliary chemical coupling agent. The final concentrations of each component were as follows: 20% polyethylene glycol diacrylate, 10% (w / v) gelatin (Cel), 0.3% (w / v) LAP, and 0.1% CMC / NHS. The specific preparation steps are as follows:

[0152] 1. Preparation of the lower layer photoinitiator solution: Prepare the lower layer LAP aqueous solution. Take this solution and add it to a sterile centrifuge tube.

[0153] 2. Gelatin Dissolution: Add gelatin powder to the centrifuge tube from step 1. Place the centrifuge tube in a water bath at 60±2℃ and heat for 25-35 minutes, manually shaking or vortexing for 10 seconds every 5 minutes until the gelatin is completely dissolved, resulting in a clear solution.

[0154] 3. Preparation of PEGDA composite solution: Accurately pipette the PEGDA700 stock solution and add it to the gelatin solution obtained in step 2. Immediately tighten the cap and vortex vigorously for 10-15 minutes to ensure that PEGDA is evenly dispersed in the gelatin solution, forming a PEGDA-gelatin composite solution (PG solution for short).

[0155] 4. Sterilization and Temporary Storage: Under weak red light or light-protected conditions, the PG solution from step 3 is sterilized by filtration through a 0.22 μm sterile filter membrane. The filtrate is collected in a brown sterile tube and immediately stored in a 4°C refrigerator protected from light. All subsequent operations must be performed under light-protected or red light-protected conditions to prevent premature photopolymerization of LAP.

[0156] 5. Preparation of the lower layer coupling agent solution: Prepare the CMC / NHS solution using the same method as the upper layer coupling agent solution.

[0157] 6. Preparation of the lower layer composite crosslinking solution: Take the PG solution prepared in step 4 into a sterile centrifuge tube. Slowly inject the CMC / NHS solution prepared in step 5 using a microsyringe, and continue stirring to mix for 1 minute.

[0158] 7. Degassing treatment: Place the mixture from step 6 in a centrifuge preheated to 37-40℃ and centrifuge at 8000-12000 rpm for 1-2 minutes to remove air bubbles.

[0159] 8. Dispensing and sealing: Transfer the degassed solution to a 4-6 mL light-protected sterile syringe. Inject the solution into a 1 mL sterile syringe through a sterile needle and seal with a sterile cap.

[0160] 9. Hot water hydrolysis treatment: Place the syringe containing the solution at room temperature (20-25℃) for 30-60 minutes to allow for initial cross-linking and the formation of a solid gel. Place the syringe containing the solution in a water bath at 90-100℃ and heat for 1-2 hours to complete the pre-cross-linking preparation of the lower gel layer. After curing, store at room temperature away from light.

[0161] (III) Layered infusion of upper and lower gel layers

[0162] An integrated biomimetic corneal gel was constructed using a two-layer infusion process.

[0163] 1. Take out the corresponding volumes of gel according to the upper gel to lower gel volume ratio of 5:5;

[0164] 2. Sequentially inject the upper and lower chambers of a specialized double-layer syringe with a removable septum;

[0165] 3. Remove the middle septum and slowly push in the syringe plunger to bring the two gel layers into contact and form a stable interface;

[0166] 4. Assemble a sterile flat-tip dispensing head and set aside.

[0167] (iv) Application methods

[0168] 1. Inject the layered gel into the corneal defect using a syringe with a flat dispensing tip, ensuring it fully fills the defect area;

[0169] 2. Cover the corneal surface with an orthokeratologist and gently press to form a curved surface on the gel that matches the curvature of the cornea;

[0170] 3. Irradiate the gel region with a light source of 365–405 nm wavelength for 1–2 min to induce a photocuring reaction and allow the gel to fully solidify;

[0171] 4. Remove the orthokeratology device to complete the corneal defect repair.

[0172] Example 2

[0173] Preparation of the upper gel: The components included 10% methacrylamide gelatin (GelMA), 5% (w / v) type I collagen, 1 mg / mL laminin (LAM, purchased from Thermo Fisher Scientific Inc.), 0.25% (w / v) photoinitiator LAP, and 0.12% (w / v) coupling agent CMC / NHS. The preparation method was the same as in Example 1. The mixed system was allowed to stand at room temperature for 15 minutes to form a preliminary cross-linked network. Then, it was transferred to a 60°C water bath for hydrolysis treatment for 9 hours to appropriately reduce the gel modulus and obtain a viscous gel precursor with good injectability.

[0174] Preparation of the lower gel layer: The components include 10% polyethylene glycol diacrylate (PEGDA700), 20% (w / v) gelatin (Cel), 0.25% (w / v) LAP, and 0.08% (w / v) CMC / NHS. The preparation method is the same as in Example 1. This mixture is first reacted in a 60°C water bath for 2 hours to complete the initial cross-linking; then it is placed in a 100°C water bath for further heat curing for 1 hour, finally forming a gel precursor with appropriate mechanical strength and maintaining plasticity.

[0175] Layered infusion: The upper and lower gel layers are injected into the independent chambers of the double-layer syringe at a volume ratio of 3:7. After removing the intermediate septum, the syringe is pushed slowly and at a constant speed to allow the two gel layers to make smooth contact at the interface, forming a stable and continuous layered structure.

[0176] Photocuring: The layered gel was injected into the corneal defect area and shaped using a molding die to match its curvature with the surrounding corneal tissue. The gel area was then irradiated with 405 nm visible light for 1 minute. Under photoinitiation, the gel system underwent free radical polymerization, ultimately curing into a single, integrated layered corneal gel with a complete structure, strong interlayer bonding, and a biomimetic morphology.

[0177] Example 3

[0178] Preparation of the upper gel: The components included 10% methacrylamide gelatin (GelMA), 20% (w / v) type I collagen, 1 mg / mL laminin (LAM, purchased from Thermo Fisher Scientific Inc.), 0.25% (w / v) photoinitiator LAP, and 0.12% (w / v) coupling agent CMC / NHS. The preparation method was the same as in Example 1. The mixed system was allowed to react at room temperature for 15 minutes to form a preliminary cross-linked network. Then, it was transferred to a 60°C water bath for hydrolysis treatment for 9 hours to appropriately reduce the gel modulus and obtain a viscous gel precursor with good injectability.

[0179] Preparation of the lower gel layer: The components include 5% chondroitin sulfate (Chsma), 20% (w / v) gelatin (Cel), 0.25% (w / v) LAP, and 0.08% (w / v) CMC / NHS. The preparation method is the same as in Example 1. This mixture is first reacted in a 60°C water bath for 2 hours to complete the initial cross-linking; then it is placed in a 100°C water bath for further heat curing for 1 hour, finally forming a gel precursor with appropriate mechanical strength and maintaining plasticity.

[0180] Layered infusion: The upper and lower gel layers are injected into the independent chambers of the double-layer syringe at a volume ratio of 3:7. After removing the intermediate septum, the syringe is pushed slowly and at a constant speed to allow the two gel layers to make smooth contact at the interface, forming a stable and continuous layered structure.

[0181] Photocuring: The layered gel was injected into the corneal defect area and shaped using a molding die to match its curvature with the surrounding corneal tissue. The gel area was then irradiated with 405 nm visible light for 1 minute. Under photoinitiation, the gel system underwent free radical polymerization, ultimately curing into a single, integrated layered corneal gel with a complete structure, strong interlayer bonding, and a biomimetic morphology.

[0182] Example 4: Preparation of a single upper gel

[0183] This embodiment uses only the upper gel formulation and preparation process to obtain a single-component corneal gel, and the specific steps are the same as in Embodiment 2.

[0184] Example 5: Preparation of a single-layer lower gel

[0185] This embodiment uses only the lower gel formulation and preparation process to obtain a single-component corneal gel, and the specific steps are the same as in Embodiment 2.

[0186] Test Example 1: Structural Feature Analysis

[0187] To verify the structural features of the bilayer hydrogel in Example 1 and the realization of the corneal biomimetic design, this study performed differential staining treatments on the upper and lower gel layers (red for the upper layer and green for the lower layer) to visually present their layered structure. In practical applications, the hydrogel provided by this invention is colorless and transparent. The results show that the interface between the two gel layers is continuous, and there is no obvious interpenetration or mixing, indicating that the layered packaging process successfully constructed a stable bilayer gel structure. Figure 1 (A). The layered gel prepared in Example 1 was then subjected to tissue staining and electron microscopy characterization. Hematoxylin-eosin staining further confirmed that the two layers had a continuous and complete interface, without obvious cracks or structural interruptions. Figure 1 (B); Scanning electron microscopy revealed that the upper gel exhibited a typical porous and loose network structure, while the lower gel had a relatively dense network structure with smaller pores, reflecting the difference in microscopic morphology between the upper and lower layers. Figure 1 (C) Transmission electron microscopy analysis further revealed that the cross-linking density of the lower gel was significantly higher than that of the upper gel, exhibiting a denser fibrous network structure and a smaller mesh size. Figure 1 (D, E). This result proves that the present invention has successfully constructed a biomimetic layered structure for the cornea, with significant differences in structure and density between the upper and lower layers.

[0188] Test Example 2: Cell Compatibility and Healing Promotion Capacity Test

[0189] Using human corneal epithelial cells and stromal cell lines as models, the two cell lines were seeded onto the surfaces of pure upper gel and pure lower gel, respectively, and cultured for 48 hours. The results were then analyzed by live / dead fluorescence staining. Figure 2 The results showed that epithelial cells and stromal cells adhered well to both gel surfaces, exhibiting normal cell morphology and continuous lamellar structure, with only a small amount of dead cell fluorescence signal. The survival rates of epithelial cells on the upper gel surface were 97.2±1.5% and stromal cells 96.8±1.2%, while those on the lower gel surface were 96.5±1.8% and stromal cells 95.9±2.0%, confirming that neither the upper nor lower gels exhibited significant cytotoxicity and possessed excellent cell compatibility. Using epithelial cells as a model, a scratch assay was conducted to detect corneal epithelial cell migration. After 36 hours of culture, the epithelial cell migration rate in the upper gel group reached 91.5±4.2%, with the scratched area filled by a large number of migrating cells, significantly narrowing the wound width. In contrast, the epithelial cell migration rate in the lower gel group was only 55.8±3.5%, with significantly weaker cell migration and filling effect (p<0.001). This demonstrates that the upper gel effectively promotes corneal epithelial cell migration and exhibits superior wound healing ability.

[0190] Test Example 3: Intraocular Pressure Tolerance Test

[0191] To evaluate the structural stability and deformation resistance of the biomimetic layered gel under physiological conditions, a simulated intraocular pressure tolerance test was conducted. Using an artificial anterior chamber model, the material was fixed in a simulated corneal location. Air was continuously injected at a constant rate using a microinfusion pump to increase the intraocular pressure until the material structure failed. The maximum intraocular pressure (IOP) it could withstand was recorded. Figure 3 ).

[0192] Test results show that the layered gel of Example 2 of this invention exhibits excellent compressive strength, with a burst pressure of 116.7 ± 14.4 mmHg. Furthermore, the material structure remains intact without significant expansion or deformation as the pressure gradually increases. In contrast, the burst pressure of the pure upper layer gel is 82.7 ± 10.6 mmHg, showing significant expansion and structural deformation under pressure. The burst pressure of the pure lower layer gel is only 45.3 ± 2.6 mmHg, indicating weaker compressive strength. The burst pressure of the biomimetic layered gel is much higher than that of a single upper or lower layer gel, indicating that the cross-linking and integration of the two gel layers provides a synergistic reinforcing effect.

[0193] The above results show that the biomimetic layered gel constructed in this invention has good mechanical stability and resistance to intraocular pressure. Its pressure tolerance is much higher than the normal physiological intraocular pressure range of human eyes (10–21 mmHg), which can provide reliable structural support for corneal tissue and meet its mechanical needs under long-term physiological environment and short-term pressure surge (such as rubbing the eyes, closing the eyes forcefully, etc.).

[0194] Test Example 4: Mechanical Property Testing of Bionic Layered Gels and Single-Component Gels

[0195] To systematically characterize the differences in mechanical properties of the biomimetic layered gel, upper gel, and lower gel of this invention, and to verify the synergistic enhancement effect of the layered structure, tensile mechanical tests were conducted using an electronic universal testing machine, referring to GB / T 16491-2018 Standard for Testing Mechanical Properties of Biomedical Polymer Materials. All samples were photocured into uniformly sized strips (20 mm long, 5 mm wide, and 1 mm thick), with 5 parallel samples set for each group, and the testing rate was 1 mm / min.

[0196] The results (Table 1) show that the pure upper gel exhibits good ductility, with an elongation at break of 65.2±5.8%, but its tensile strength and elastic modulus are relatively low, at 7.8±0.9 kPa and 0.12±0.02 MPa, respectively, and its toughness is 0.8±0.1 MJ / m³, indicating weak mechanical support and resistance to breakage. The lower gel has significantly better tensile strength and elastic modulus than the upper gel, at 24.5±2.1 kPa and 0.85±0.07 MPa, respectively, and its toughness reaches 5.2±0.4 MJ / m³, indicating strong resistance to breakage, but its ductility is relatively low. The lower gel has poor extensibility, with an elongation at break of only 18.3±2.4%, making it prone to brittle fracture. The biomimetic layered gel achieves complementary performance between the two single-component gels, with a tensile strength of 29.6±3.2 kPa, an elastic modulus of 0.92±0.08 MPa, and improved toughness of 7.5±0.6 MJ / m³, while maintaining good extensibility and an elongation at break of 42.5±4.7%. It possesses the high strength, high modulus, and high toughness of the lower gel, while also taking into account the good extensibility of the upper gel. Its mechanical properties are more suitable for the mechanical requirements of the corneal physiological environment.

[0197] Table 1

[0198] Example Tensile strength (kPa) Extensibility (%) Elastic modulus (MPa) Toughness (MJ / m³) 2 29.6±3.2 42.5±4.7 0.92±0.08 7.5±0.6 3 24.7±2.1 37.8±5.0 0.85±0.12 6.8±0.4 4 7.8±0.9 65.2±5.8 0.12±0.02 0.8±0.1 5 24.5±2.1 18.3±2.4 0.85±0.07 5.2±0.4

[0199] Test Example 5: Evaluation of In Vivo Corneal Lamellar Defect Repair Effect

[0200] To systematically evaluate the repair effects of different filler materials in vivo, this study established a rabbit corneal lamellar defect model and conducted a 4-week comparative study on the repair effects. Eighteen healthy New Zealand white rabbits were randomly divided into three groups (n=6 per group), using Example 2 (bionic layered gel), Example 4 (upper layer gel), and Example 5 (lower layer gel) as filler materials for repair, respectively. A lamellar defect with a diameter of 3.5 mm and a depth of 200 μm was created in the center of the rabbit cornea. After injecting each material into the defect cavity, in-situ irradiation with blue light at a wavelength of 405 nm and a power density of 10 mW / cm² was performed for 60 seconds.

[0201] On postoperative days 7 and 28, corneal transparency, epithelial integrity, and gross healing were observed using a slit-lamp microscope. Optical coherence tomography (OCT) was used to assess structural repair, material retention, and dynamic changes in thickness in the defect area.

[0202] General observation results:

[0203] On the 7th day after surgery, the corneal epithelium in both Example 2 and Example 4 groups had basically healed, the wound surface was smooth, and no obvious infection or acute rejection reaction was observed. Figure 4(A) By the 28th day after surgery, the cornea in Group 2 maintained good transparency, the epithelial layer was continuous and intact, and no obvious scars or neovascularization were observed; the cornea in Group 4 also maintained good transparency and the epithelial layer was intact; while no corneal epithelial regeneration was observed in Group 5, and persistent defects remained on the surface.

[0204] OCT structural analysis:

[0205] OCT results ( Figure 4 As shown in Figure B), the material in Example 2 maintained a complete structure and stable thickness within the defect area, with a continuous epithelial layer covering the surface, and natural integration at the material-host interface. The material in Example 4 showed a significant decrease in thickness at 28 days post-surgery, indicating significant degradation. Although the material in Example 5 remained within the defect area, its surface lacked complete epithelial coverage.

[0206] Histological analysis:

[0207] To further evaluate the repair effect and material degradation behavior at the microscopic level, corneal tissue was obtained 4 weeks postoperatively for hematoxylin-eosin staining. In Example 2, the defect area was shown to be fully filled with the material, with a full contour and a surface tightly covered by stratified epithelial cells. The internal structure of the material remained stable, and no obvious signs of inflammatory cell infiltration or degradation were observed. Figure 4 (C) In Example 4, only a small amount of residual gel structure was visible in the original defect area, indicating that the material had undergone rapid and widespread degradation and could no longer provide long-term mechanical support for the cornea. In Example 5, the material was still visible in the defect area and maintained a relatively good thickness, but the surface continued to lack epithelial coverage. Long-term incomplete epithelium will increase the risk of material exposure, infection, and rejection.

[0208] Conclusion: The in vivo experimental results demonstrate that the biomimetic layered gel provided by this invention exhibits excellent long-term structural stability, degradation resistance, and good epithelial integration in the repair of lamellar corneal defects in rabbits, effectively maintaining the morphological integrity and optical transparency of the repaired area. The upper layer material alone, due to its excessively rapid degradation, failed to provide the necessary structural support, leading to tissue collapse after repair. While the lower layer material alone possessed some structural stability, its lack of complete epithelial coverage hindered long-term biointegration and functional recovery. This study confirms that this invention possesses superior long-term repair potential in corneal tissue engineering.

[0209] The specific embodiments described above do not constitute a limitation on the scope of protection of this invention. Those skilled in the art should understand that various modifications, combinations, sub-combinations, and substitutions can be made based on the inventive principles and other factors. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this invention should be included within the scope of protection of this invention.

Claims

1. A biomimetic layered gel, characterized in that, It includes an upper gel and a lower gel; wherein the upper gel contains methacrylamide gelatin, collagen, laminin, photoinitiator, and coupling agent; The lower gel contains polyethylene glycol diacrylate or methacrylamide sulfate cartilage, as well as gelatin, photoinitiator, and coupling agent; The upper and lower gels each have a three-dimensional cross-linked network; the upper gel has a porous and loose network structure, while the lower gel has a fibrous network structure. The fluid upper and lower gels are combined in a volume ratio of 1:9 to 5:5, and the two gels form an integrated structure through cross-linked transition zones. The pore size of the lower gel network is smaller than that of the upper gel network; In the upper gel, the concentration of methacrylamide gelatin is 10%-20% (w / v), the concentration of collagen is 5%-20% (w / v), the concentration of laminin is 0.8 mg / mL-1.2 mg / mL, the concentration of photoinitiator is 0.2%-0.3%, and the concentration of coupling agent is 0.05%-0.2%. In the lower gel, the concentration of polyethylene glycol diacrylate or methacrylamide sulfate cartilage is 5%-20%, the concentration of gelatin is 5%-10%, the concentration of photoinitiator is 0.2%-0.3%, and the concentration of coupling agent is 0.1%-0.5%.

2. The biomimetic layered gel as described in claim 1, characterized in that, The cross-linking density of the lower gel is higher than that of the upper gel.

3. The biomimetic layered gel as described in claim 1, characterized in that, The biomimetic layered gel has an overall transmittance of ≥80%, a refractive index of 1.33-1.40, and a tensile strength of ≥20kPa.

4. The method for preparing the biomimetic layered gel as described in any one of claims 1-3, characterized in that, Includes the following steps: a) Prepare the upper layer composite crosslinking solution and the lower layer composite crosslinking solution; The upper composite cross-linking solution includes 10%-20% (w / v) methacrylamide gelatin, 5%-20% (w / v) collagen, 0.8 mg / mL-1.2 mg / mL laminin, 0.2%-0.3% photoinitiator, and 0.05%-0.2% coupling agent. The lower-layer composite crosslinking solution includes 5%-20% polyethylene glycol diacrylate or chondroitin sulfate with methacrylamide, 5%-10% (w / v) gelatin, 0.2%-0.3% photoinitiator, and 0.1%-0.5% coupling agent; b) After the upper composite crosslinking solution and the lower composite crosslinking solution react separately, an upper solid gel and a lower solid gel with a three-dimensional crosslinking network are obtained; c) The upper solid gel and the lower solid gel are subjected to hydrothermal treatment to obtain fluid upper gel and lower gel; d) Combine the upper and lower gels in a volume ratio of 1:9 to 5:5 to bring the two gels into contact and cross-link them, thus obtaining a biomimetic layered gel.

5. The method for preparing the biomimetic layered gel as described in claim 4, characterized in that, The collagen materials include gelatin, type I / III collagen, and recombinant human collagen.

6. The method for preparing the biomimetic layered gel as described in claim 4, characterized in that, The photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphonate, sodium phenyl-2,4,6-trimethylbenzoylphosphonate, or a combination thereof.

7. The method for preparing the biomimetic layered gel as described in claim 4, characterized in that, The coupling agent is a combination of a carbodiimide and an N-hydroxy compound, wherein the carbodiimide is selected from one or more of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, and 1-cyclohexyl-2-morpholinoethylcarbodiimide; and the N-hydroxy compound is selected from one or more of N-hydroxysuccinimide and sulfonyl-N-hydroxysuccinimide. Alternatively, the coupling agent may be a triazine-onium salt coupling agent, including 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium salt; Alternatively, the coupling agent may be a ureonium / phosphoniumium salt coupling agent selected from one or more of the following: O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate, benzotriazol-1-yl-oxytripyrrolylphosphine hexafluorophosphate, or (benzotriazol-1-yloxy)tri(dimethylamino)phosphonium hexafluorophosphate.

8. The method for preparing the biomimetic layered gel as described in claim 4, characterized in that, The molecular weight of polyethylene glycol diacrylate is 500-10000.

9. The method for preparing the biomimetic layered gel as described in claim 4, characterized in that, In step b), the reaction conditions are settling at 10–34°C.

10. The method for preparing the biomimetic layered gel as described in claim 4, characterized in that, In step c), the hydrothermal treatment is a water bath at 40-150°C.

11. The method for preparing the biomimetic layered gel as described in claim 4, characterized in that, The viscosity of the upper gel and the lower gel is 2000-10,000 mPa·s.

12. The use of the biomimetic layered gel as described in any one of claims 1-3 in the preparation of a medical device for repairing corneal damage.

13. The application as described in claim 12, characterized in that, The corneal injuries include ocular surface injuries, corneal epithelial injuries, corneal stromal injuries, corneal ulcers, corneal perforations, and corneal lacerations.

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