Ultra-low swelling artificial cornea imitating natural corneal fiber arrangement and preparation method

Abstract

The present invention provides an ultra-low swelling artificial cornea that mimics the fiber arrangement of a natural cornea and a preparation method, belonging to the technical field of biomedical materials. The present invention dissolves type I collagen in PBS buffer, adds a photofunctionalized active agent for chemical modification, stirs in the dark, and then freeze-dries to obtain photofunctionalized collagen; the photofunctionalized collagen is dissolved, and then a collagen fiber membrane is prepared by electrospinning; the bio-ink and the collagen fiber membrane are subjected to phase change DLP 3D bioprinting to obtain an artificial cornea with a collagen fiber sandwich structure. The present invention achieves multi-layer arrangement through DLP 3D bioprinting, mimicking the fiber orientation of a natural cornea and significantly improving mechanical anisotropy; the composite phase change DLP 3D printing method of GelMA / HA-DN and photocrosslinked collagen fibers makes it have good manufacturability, maintains high transmittance, and maintains high mechanical strength.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and in particular relates to an ultra-low swelling artificial cornea imitating the arrangement of natural corneal fibers and a preparation method thereof. Background Art

[0002] An artificial cornea refers to a special refractive device made of artificial synthetic materials. It is used to replace the cloudy cornea that blocks the optical pathway of the eye after a disease, so that the patient can regain a certain degree of vision.

[0003] The current mainstream artificial corneal materials include polymers (such as the Boston-type artificial cornea of ​​polymethyl methacrylate (PMMA) / PHEMA), hydrogels (such as polyethylene glycol derivatives), and biomaterials (such as decellularized corneal stroma). Among them, synthetic polymer materials and hydrogel materials have stable and controllable mechanical properties, but poor biocompatibility, which can cause severe wear or corneal ablation in the later stage. In addition, they lack biomimetic fiber arrangement and cannot integrate with host tissues. Natural biomaterials are similar to natural tissues in composition and have good biocompatibility, but they have poor mechanical properties, complex processes, difficulty in restoring light transmittance, and the risk of immune rejection. In addition, electrospinning technology can also be used to prepare collagen fiber membranes, but the fiber arrangement of collagen fiber membranes is mostly disordered or unidirectional, lacking a biomimetic orthogonal structural design.

[0004] It can be seen that the common problems of the above-mentioned artificial cornea materials are: (1) high swelling rate. For example, hydrogel materials swell significantly after absorbing water (such as pure GelMA), which leads to deterioration of transmittance and mechanical properties, affecting long-term stability; (2) insufficient mechanical strength. The disordered or single-oriented fiber structure is difficult to simulate the mechanical anisotropy of the natural cornea, resulting in insufficient tear resistance and deformation resistance; (3) the contradiction between transmittance and biomimetic properties. The traditional method increases the transmittance by increasing the density of the material, but sacrifices the biomimetic properties of the fiber arrangement, affecting cell migration and integration.

[0005] Currently, the application of tissue engineering in the preparation of artificial corneas has important scientific significance and clinical value. Its core lies in breaking through the limitations of traditional artificial corneas through the combination of biomimetic design, biomaterials and cell technology, and providing innovative solutions to solve problems such as corneal donor shortages, postoperative complications and functional recovery. In the existing technology, tissue engineering corneal scaffolds mostly use acellular matrices or collagen scaffolds combined with cell culture. For example, patent CN119139549A uses electric field-triggered dynamic assembly of collagen fiber membranes, but only relies on a single riboflavin cross-linking natural collagen component without introducing other high-bond energy synergistic cross-linking. This method makes it difficult to simultaneously control the swelling rate and mechanical properties, resulting in the material being easily degraded or deformed in the body fluid environment and poor cross-linking stability. At the same time, existing strategies cannot achieve a sandwich structure with multiple layers of orthogonal fiber arrangement, the structural complexity is low, and it is difficult to simulate the layered microstructure of the natural cornea. Patent CN117899266A mentions the use of intelligent manufacturing methods to prepare orthogonal grids, which can obtain artificial corneas with a certain collagen fiber microstructure. However, the cross-linking method of this structure is uncertain and its stability needs to be investigated. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide an ultra-low swelling artificial cornea that mimics the fiber arrangement of the natural cornea and a preparation method. Through the collaborative design of collagen photocross-linking modification and multi-layer collagen fiber sandwich structure based on phase change DLP 3D bioprinting, combined with light curing technology and the physical phase change properties of thermosensitive materials, by dynamically controlling the cross-linking method (photochemical cross-linking and physical cross-linking in coordination), efficient and high-precision preparation of complex structures is achieved, solving the technical problems in the existing technology of low mechanical strength and decreased transmittance of artificial cornea caused by high material swelling rate, insufficient biomimetic fiber arrangement and poor cross-linking stability.

[0007] To solve the above technical problems, the present invention adopts a technical solution: a method for preparing an ultra-low swelling artificial cornea that mimics the arrangement of natural corneal fibers, comprising the following steps:

[0008] S1. Preparation of photofunctionalized collagen: Dissolve type I collagen in phosphate buffer, add active reagent for photofunctionalization modification for chemical modification, stir in the dark, and freeze-dry to obtain photofunctionalized collagen (photoresponsive collagen).

[0009] Furthermore, in step S1, the concentration of the phosphate buffer is 0.5% to 4%.

[0010] Furthermore, in step S1, the amount of the active agent added for photofunctionalization modification is 2% to 10% of the mass of type I collagen, and the active agent for photofunctionalization modification is methacrylic anhydride (MA) or N-hydroxysuccinimide acrylate (NHS-acrylate) containing a C=C functional group.

[0011] Furthermore, in step S1, stirring is performed at room temperature (25-37° C.) in the dark for 6-12 hours; and then freeze-drying is performed at -50--80° C. and a vacuum degree of less than or equal to 1 Pa for 18 hours.

[0012] The above steps endow collagen fibers with photocuring ability by introducing photocrosslinking groups (such as C=C double bonds), enabling them to form stable chemical crosslinks with bio-ink in subsequent DLP printing, inhibiting swelling and enhancing interfacial bonding strength.

[0013] S2. Preparation of collagen fiber membrane: dissolving the photofunctionalized collagen from step S1, and then preparing collagen fiber membrane by electrospinning.

[0014] Furthermore, the collagen fiber membrane includes an ordered collagen fiber membrane and a random collagen fiber membrane.

[0015] Furthermore, in step S2, the photofunctionalized collagen is dissolved in hexafluoroisopropanol (HFIP) or an acetic acid / water mixed solvent (the volume ratio of acetic acid to water is 8:2).

[0016] Furthermore, in step S2, the electrospinning parameters are: voltage 15-40 kV, spinning solution flow rate 0.5-2 ml / h, receiving distance 10-30 cm, and receiving by a rotating receiving device with a rotation speed of 500-5000 rpm.

[0017] S3. Phase change DLP 3D printing to construct collagen fiber sandwich structure.

[0018] S31. Prepare bio-ink for phase change DLP 3D printing: mix methacrylated gelatin (GelMA), HA-DN and photoinitiator to form a temperature-sensitive liquid bio-ink.

[0019] Furthermore, in step S31, the concentration of GelMA is 5% to 15% (w / v). GelMA is a thermosensitive phase-change material that achieves temporary physical crosslinking support through temperature regulation (gelation at 25°C, liquid at 37°C). HA-DN is prepared by mixing aldehyde-modified hyaluronic acid (HA-CHO) and hydrazide-modified hyaluronic acid (HA-ADH). The mass ratio of HA-CHO to HA-ADH is 1:1 to 1:3. The hyaluronic acid is modified with aldehyde (-CHO) and hydrazide (-ADH) groups, forming dynamic covalent bonds (Schiff base bonds), which impart tear resistance and self-healing properties to the material. The concentration of the photoinitiator LAP is 0.1% to 0.5%. LAP is a 405 nm wavelength-sensitive initiator that reduces UV damage to biological activity.

[0020] S32. Constructing a collagen fiber sandwich structure layer by layer: performing phase change DLP 3D bioprinting using the bio-ink prepared in step S31 and the collagen fiber membrane prepared in step S2 to obtain a collagen fiber sandwich structure.

[0021] Furthermore, the printing process includes the following steps:

[0022] S321. Preheating the bio-ink: Heat the bio-ink to 37-45°C to ensure good fluidity. Use a scraper with a high-precision displacement system to coat the ink on the printing platform to form a uniform ultra-thin layer with a thickness of 10-50 μm.

[0023] S322, low-temperature gelation: cooling to below 25°C (preferably 15-20°C), GelMA is rapidly physically cross-linked through hydrogen bonds to form a stable support, achieving physical gelation to obtain a gel layer;

[0024] S323, collagen fiber membrane laying: attaching the collagen fiber membrane to the surface of the gel layer prepared in step S322, and fixing the position of the collagen fiber membrane by surface tension to achieve a tight fit;

[0025] S324, In-situ patterned photocrosslinking: Using the DMD in the DLP 3D printing system for pattern projection (parameters: wavelength 405nm, light intensity 10~50mW / cm 2 , exposure time 5-30s) to solidify the current layer, so that the collagen fiber membrane and the bio-ink layer are chemically cross-linked, that is, the photofunctionalized collagen fibers of the collagen fiber membrane and the GelMA / HA-DN layer of the bio-ink form a chemical cross-linking network, thereby increasing the interface strength;

[0026] S325, alternating stacking: repeat steps S321 to S324 to arrange the next layer of collagen fiber membrane, and finally form a multi-layer collagen fiber sandwich structure.

[0027] In step S3, the collagen fiber sandwich structure includes an orthogonal collagen fiber sandwich structure, a unidirectional collagen fiber sandwich structure and a random collagen fiber sandwich structure.

[0028] S4. The collagen fiber sandwich structure formed in step S3 is reheated to 37°C and maintained for 5-10 minutes. The bio-ink (GelMA / HA) in the non-light-projected area is restored to liquid state. The uncross-linked bio-ink is gently rinsed with PBS solution to obtain an artificial cornea with a stable collagen fiber sandwich structure.

[0029] Another object of the present application is to provide an artificial cornea prepared by a method for preparing an ultra-low swelling artificial cornea that mimics the natural corneal fiber arrangement.

[0030] Furthermore, the number of layers of the artificial cornea collagen fiber sandwich structure is 3 to 20.

[0031] Furthermore, the swelling rate of the artificial cornea is no higher than 10%, and the transmittance is greater than 85%.

[0032] The beneficial effects of the present invention are:

[0033] This invention provides a low-swelling artificial cornea with a collagen fiber sandwich structure and its preparation method. By integrating photofunctionalized collagen fibers, electrospinning technology, and phase-change DLP 3D bioprinting, the invention addresses existing problems with artificial corneas, such as high swelling rate, insufficient mechanical strength, and the lack of biomimetic structures, resulting in reduced light transmittance and poor biocompatibility. This invention can also be applied to other biomimetic layered biomedical devices, such as heart valves, cartilage, and skin scaffolds.

[0034] (1) The collagen fibers of the present invention are photofunctionalized and then arranged in multiple layers using DLP 3D bioprinting, mimicking the fiber orientation of the natural cornea and significantly enhancing mechanical anisotropy (increased tensile strength). The composite phase-change DLP 3D printing of GelMA / HA-DN and photocrosslinked collagen fibers enables good manufacturability, maintaining high light transmittance (>90%), and high mechanical strength.

[0035] (2) The biomimetic microstructure of the present invention and its compatibility with surgery: The sandwich design (fiber membrane-hydrogel-fiber membrane) enhances suture stability, inhibits swelling rate (ultra-low swelling), and reduces the risk of postoperative graft displacement. The orthogonally arranged fiber network promotes directional cell migration, accelerates integration with the host cornea, and reduces rejection reactions. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The present invention will be described in detail below with reference to the accompanying drawings and in combination with examples, and the advantages and implementation modes of the present invention will become more apparent. The contents shown in the accompanying drawings are only used to illustrate the present invention and do not constitute any limitation to the present invention. In the accompanying drawings:

[0037] Figure 1 This is the SEM image of the electrospun ordered collagen fiber membrane of the present invention.

[0038] Figure 2 This is a contact angle test diagram of the collagen fiber membrane of the present invention.

[0039] Figure 3 This is a rheological test diagram of the bio-ink of the present invention.

[0040] Figure 4 This is a physical picture of the self-healing performance of the bio-ink of the present invention.

[0041] Figure 5 This is a mechanical test diagram of the artificial cornea of ​​the present invention.

[0042] Figure 6 This is a diagram showing the swelling rate of the artificial cornea of ​​the present invention.

[0043] Figure 7 This is a light transmittance diagram of the artificial cornea of ​​the present invention.

[0044] Figure 8 This is a biocompatibility test diagram of the artificial cornea of ​​the present invention.

[0045] Figure 9 This is a diagram showing the cell life and death status on the surface of the artificial cornea of ​​the present invention.

[0046] Figure 10 This is a diagram showing the cytoskeleton expression of the artificial cornea of ​​the present invention. DETAILED DESCRIPTION

[0047] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is further described in detail below in conjunction with the best embodiment.

[0048] A method for preparing an ultra-low swelling artificial cornea that mimics the arrangement of natural corneal fibers comprises the following steps:

[0049] S1. Preparation of photofunctionalized collagen: Dissolve type I collagen in phosphate buffered saline (PBS) at a concentration of 0.5% to 4%, add methacrylic anhydride (MA) or N-hydroxysuccinimide acrylate (NHS-acrylate) containing a C=C functional group as an active agent for photofunctionalization, and chemically modify the collagen. Stir in the dark at room temperature (25-37°C) for 6-12 hours. Then, freeze-dry at -50--80°C and a vacuum of 1 Pa or less for 18 hours to obtain photofunctionalized collagen (photoresponsive collagen).

[0050] The amount of active agent added for photofunctional modification is 2% to 10% of the mass of type I collagen.

[0051] The above steps endow collagen fibers with photocuring ability by introducing photocrosslinking groups (such as C=C double bonds), enabling them to form stable chemical crosslinks with bio-ink in subsequent DLP printing, inhibiting swelling and enhancing interfacial bonding strength.

[0052] S2. Preparation of collagen fiber membrane: The photofunctionalized collagen from step S1 was dissolved in hexafluoroisopropanol (HFIP) or an acetic acid / water mixed solvent (the volume ratio of acetic acid to water was 8:2), and then the collagen fiber membrane was prepared by electrospinning technology.

[0053] The collagen fiber membrane includes ordered collagen fiber membrane and random collagen fiber membrane.

[0054] The electrospinning parameters for preparing ordered collagen fiber membranes are: voltage 15~25 kV, spinning solution flow rate 0.5~2 ml / h, receiving distance 10~20 cm, received by a rotating receiving device (high-speed rotating drum) with a rotation speed of 2000~5000 rpm, or controlled by a directional electric field.

[0055] Highly oriented collagen fiber membranes (fiber diameter 50~500 nm) were prepared by directional spinning technology to simulate the natural ordered fiber arrangement.

[0056] The electrospinning parameters for preparing random collagen fiber membranes were as follows: voltage 15–40 kV, spinning solution flow rate 0.5–2 ml / h, and receiving distance 25 cm, received by a rotating receiving device (low-speed rotating drum) with a rotation speed of 500 rpm.

[0057] S3. Phase change DLP 3D printing: Phase change DLP 3D printing constructs collagen fiber sandwich structures.

[0058] S31. Prepare bio-ink for phase change DLP 3D printing: mix methacrylated gelatin (GelMA), HA-DN and photoinitiator to form a temperature-sensitive liquid bio-ink.

[0059] The concentration of GelMA is 5% to 15% (w / v). GelMA is a thermosensitive phase-change material that achieves temporary physical crosslinking through temperature control (gelation at 25°C, liquid at 37°C). HA-DN is a mixture of aldehyde-modified hyaluronic acid (HA-CHO) and hydrazide-modified hyaluronic acid (HA-ADH), with a mass ratio of HA-CHO to HA-ADH of 1:1 to 1:3. Hyaluronic acid is modified with aldehyde (-CHO) and hydrazide (-ADH) groups, forming dynamic covalent bonds (Schiff base bonds) that impart tear resistance and self-healing properties to the material. The concentration of the photoinitiator LAP is 0.1% to 0.5%. LAP is a 405 nm wavelength-sensitive initiator that reduces UV damage to bioactivity.

[0060] S32, constructing a collagen fiber sandwich structure layer by layer: using the bio-ink prepared in step S31 and the collagen fiber membrane prepared in step S2 to perform phase change DLP 3D bio-printing, the printing process includes the following steps:

[0061] S321, Bio-ink preheating: Heat the bio-ink to 37-45°C to ensure good fluidity. Use a scraper with a high-precision displacement system to coat it on the printing platform to form a uniform ultra-thin layer with a thickness of 10-50μm.

[0062] S322, low-temperature gelation: cooling to below 25°C (preferably 15-20°C), GelMA is rapidly physically cross-linked through hydrogen bonds to form a stable support, achieving physical gelation to obtain a gel layer;

[0063] S323, collagen fiber membrane laying: attaching the collagen fiber membrane to the surface of the gel layer prepared in step S322, and fixing the position of the collagen fiber membrane by surface tension to achieve a tight fit;

[0064] S324, In-situ patterned photocrosslinking: Using the DMD in the DLP 3D printing system for pattern projection (parameters: wavelength 405nm, light intensity 10~50mW / cm 2 , exposure time 5-30s) to solidify the current layer, so that the collagen fiber membrane and the bio-ink layer are chemically cross-linked, that is, the photofunctionalized collagen fibers of the collagen fiber membrane and the GelMA / HA-DN layer of the bio-ink form a chemical cross-linking network, thereby increasing the interface strength;

[0065] S325, Alternate Stacking:

[0066] Orthogonal collagen fiber sandwich structure: using an ordered collagen fiber membrane, repeat steps S321 to S324, and arrange the next layer of ordered collagen fiber membrane in a direction orthogonal to the attachment direction of step S323 (for example, if the attachment direction in step S323 is the X-axis direction, the attachment direction of the next layer of ordered collagen fiber membrane is the Y-axis direction), ultimately forming a multi-layer orthogonal collagen fiber sandwich structure (total number of layers: 3 to 20);

[0067] Unidirectional collagen fiber sandwich structure: using an ordered collagen fiber membrane, repeat steps S321 to S324, and arrange the next layer of ordered collagen fiber membrane in the same direction as the attachment direction in step S323 (e.g., if the attachment direction in step S323 is the X-axis direction, the attachment direction of the next layer of ordered collagen fiber membrane is also the X-axis direction), ultimately forming a multi-layer unidirectional collagen fiber sandwich structure (total number of layers: 3 to 20);

[0068] Random collagen fiber sandwich structure: using a random collagen fiber membrane, repeating steps S321 to S324, and randomly attaching the next layer of random collagen fiber membrane to eventually form a multi-layer random collagen fiber sandwich structure (total number of layers: 3 to 20);

[0069] S4. The collagen fiber sandwich structure formed in step S3 is reheated to 37°C and maintained for 5-10 minutes. The bio-ink (GelMA / HA) in the non-light-projected area is restored to liquid state. The uncross-linked bio-ink is gently rinsed with PBS solution to obtain an artificial cornea with a stable collagen fiber sandwich structure.

[0070] The collagen fiber sandwich structure prepared using this method effectively mimics the mechanical anisotropy of the natural cornea, improving overall tensile strength. Furthermore, the enhanced interfacial interactions limit water penetration, and combined with the hydrophobic effect of the HA dynamic bonds, the swelling rate is reduced to below 10%. Furthermore, the orderly fiber arrangement and photocrosslinking effectively stabilize the fiber layer while reducing light scattering, optimizing the transmittance of the artificial cornea.

[0071] The present invention achieves the coordinated optimization of ultra-low swelling, high transmittance and bionic mechanical properties of the artificial cornea through the full-process innovation of photofunctionalization modification-electrospinning orientation-phase change DLP printing, filling the gap in traditional technologies where material properties and structural bionics are difficult to balance.

[0072] Another object of the present application is to provide an artificial cornea prepared by a method for preparing an ultra-low swelling artificial cornea that mimics the natural corneal fiber arrangement.

[0073] The number of layers of the artificial cornea collagen fiber sandwich structure is 3 to 20, wherein the angle between adjacent fiber layers of the orthogonal collagen fiber sandwich structure is 90°±5°.

[0074] The swelling rate of the artificial cornea is no more than 10%, and the transmittance is greater than 85%.

[0075] Example:

[0076] A method for preparing an ultra-low swelling artificial cornea that mimics the arrangement of natural corneal fibers comprises the following steps:

[0077] S1. Preparation of photofunctionalized collagen: Dissolve type I collagen in 2% PBS buffer and chemically modify it by adding 10% N-hydroxysuccinimide acrylate to the type I collagen. Stir in the dark for 6 h at room temperature. Then, freeze-dry in a freeze dryer at -70°C and a vacuum of 0.8 Pa for 18 h to obtain a white porous sponge-like photofunctionalized collagen (photoresponsive collagen).

[0078] S2. Preparation of ordered collagen fiber membrane: The photofunctionalized collagen prepared in step S1 was dissolved in 8% HFIP and loaded into a 20 ml syringe. The electrospinning parameters were set as follows: voltage 20 kV, spinning solution flow rate 1 ml / h, receiving distance 15 cm, and the membrane was received by a high-speed rotating drum at a speed of 3000 rpm. The spinning lasted for 2 h. Figure 1 As shown, an ordered collagen fiber membrane with a fiber diameter of about 200 nm and unidirectional arrangement was obtained.

[0079] like Figure 2As shown in the figure, the contact angle test was performed and the contact angle of the prepared collagen fiber membrane was 35° (the bio-ink layer was 60°), indicating that the cross-linked collagen fibers were more hydrophilic and conducive to cell adhesion.

[0080] Preparation of random collagen fiber membrane: The photofunctionalized collagen from step S1 was dissolved in 8% HFIP and loaded into a 20 ml syringe. The electrospinning parameters were set as follows: voltage 20 kV, spinning solution flow rate 1 ml / h, receiving distance 25 cm, and received by a low-speed rotating drum at a speed of 500 rpm. The spinning lasted for 2 h.

[0081] S3. Phase change DLP 3D printing: Phase change DLP 3D printing constructs collagen fiber sandwich structures.

[0082] S31. Prepare bio-ink for phase-change DLP 3D printing: dissolve 10% GelMA, 0.3% HA-CHO, 0.8% HA-ADH (the mass ratio of HA-CHO to HA-ADH is 1:1), and 0.3% LAP in PBS. Stir at 37°C for 2 h until completely dissolved to form a thermosensitive liquid bio-ink.

[0083] like Figure 3 and Figure 4 As shown, Figure 3 (a) is a frequency measurement chart. A frequency sweep (0.1–10 Hz) shows that the storage modulus (G') of the bioink containing HA-DN (experimental groups 1 and 2, with the HA-DN concentration in group 2 being higher than that in group 1) significantly increased (compared to the control group, which was pure GelMA). Furthermore, further increases in HA-DN concentration had a limited effect on the storage modulus. Figure 3 (b) shows a photocuring test. Under 405nm light, the bioink's composite viscosity significantly increased within 30 seconds, demonstrating its photo-controlled polymerization capability. After photocuring, the composite viscosity increased with increasing HA-DN concentration. Combined with the results of the frequency conversion experiment, the optimal HA-DN concentration was 0.3%, which was used in subsequent experiments.

[0084] like Figure 4 As shown in the figure, the cut bio-ink gel was able to heal together and had a certain tear resistance after being left at room temperature for 30 minutes, indicating that the bio-ink layer used formed dynamic covalent bonds and had good self-healing properties.

[0085] S32, constructing a collagen fiber sandwich structure layer by layer: using the bio-ink prepared in step S31 and the collagen fiber membrane prepared in step S2 to perform phase change DLP 3D bio-printing, the printing process includes the following steps:

[0086] S321. Preheating of bio-ink: Heat the bio-ink to 37°C to ensure good fluidity. Use a scraper with a high-precision displacement system to coat it on the printing platform to form a uniform ultra-thin layer with a thickness of 50 μm.

[0087] S322, low-temperature gelation: When the temperature is lowered to below 18°C, GelMA is rapidly physically cross-linked through hydrogen bonds to form a stable support, achieving physical gelation and obtaining a gel layer;

[0088] S323, oriented lamination of collagen fiber membrane: attaching the collagen fiber membrane to the surface of the gel layer prepared in step S322, and fixing the position of the collagen fiber membrane by surface tension to achieve close adhesion;

[0089] S324, In-situ patterned photocrosslinking: Using the DMD in the DLP 3D printing system for pattern projection (parameters: wavelength 405nm, light intensity 25mW / cm 2 , exposure time 15s) to solidify the current layer, so that the collagen fiber membrane and the bio-ink layer are chemically cross-linked, that is, the photofunctionalized collagen fibers of the collagen fiber membrane and the GelMA / HA-DN layer of the bio-ink form a chemical cross-linking network, thereby increasing the interface strength;

[0090] S325, Alternate Stacking:

[0091] Experimental group a: unidirectional collagen fiber sandwich structure: using an ordered collagen fiber membrane, repeat steps S321 to S324, and arrange the next layer of ordered collagen fiber membrane in the same direction as the attachment direction in step S323, ultimately forming 10 layers of unidirectional collagen fiber sandwich structure hydrogel (total thickness approximately 500 μm);

[0092] Experimental group b: orthogonal collagen fiber sandwich structure: using an ordered collagen fiber membrane, repeat steps S321 to S324, and arrange the next layer of ordered collagen fiber membrane in a direction orthogonal to the attachment direction of step S323, ultimately forming a 10-layer orthogonal collagen fiber sandwich structure hydrogel (total thickness of approximately 500 μm);

[0093] Experimental group c: random collagen fiber sandwich structure: using a random collagen fiber membrane, repeat steps S321 to S324, and randomly attach the next layer of random collagen fiber membrane to form a 10-layer random collagen fiber sandwich structure hydrogel (total thickness of about 500 μm);

[0094] The blank control group did not add collagen fiber membrane, and only printed the bio-ink layer.

[0095] S4. Rewarm the experimental group a, experimental group b, and blank control group formed in step S3 to 37°C and maintain for 5-10 minutes. The bio-ink (GelMA / HA) in the non-light-projected area is restored to liquid state. The uncross-linked bio-ink is gently rinsed with PBS solution to obtain artificial corneas.

[0096] like Figure 5 As shown, the mechanical properties of the above artificial cornea are compressed and stretched. Figure 5 In the compression performance test diagram (a), the maximum compression modulus of the orthogonal collagen fiber sandwich structure in experimental group b reached 693.3 kPa, the maximum compression modulus of the unidirectional collagen fiber sandwich structure in experimental group a was 633.5 kPa, and the maximum compression modulus of the fiber-free structure in the blank control group was 120.7 kPa. The maximum compression modulus of the random collagen fiber sandwich structure in experimental group c, while lower than those in experimental groups a and b, was still significantly higher than that in the blank control group. This shows that collagen fiber sandwich structures possess excellent compression properties, with the orthogonal collagen fiber sandwich structure achieving the best performance. Figure 5 In the compression performance test diagram (b), the compression fracture strain of the orthogonal collagen fiber sandwich structure in experimental group b reached 94.5%, the compression fracture strain of the unidirectional collagen fiber sandwich structure in experimental group a was 79%, and the compression fracture strain of the fiber-free structure in the blank control group was 60.5%. The compression fracture strain of the random collagen fiber sandwich structure in experimental group c, while lower than that of experimental group b, was still higher than that of the blank control group. This shows that collagen fiber sandwich structures have excellent compression fracture strain performance, with the orthogonal collagen fiber sandwich structure having the best performance. Figure 5 In the tensile properties test (c), the maximum tensile modulus of the orthogonal collagen fiber sandwich structure in experimental group b reached 656.2 kPa, the maximum tensile modulus of the unidirectional collagen fiber sandwich structure in experimental group a was 452.8 kPa, and the maximum tensile modulus of the fiber-free structure in the blank control group was 94.05 kPa. While the maximum tensile modulus of the random collagen fiber sandwich structure in experimental group c was lower than that of experimental groups a and b, it was still significantly higher than that of the blank control group. This indicates that collagen fiber sandwich structures possess excellent tensile properties, with the orthogonal collagen fiber sandwich structure exhibiting the best tensile properties. Figure 5 In the tensile performance test diagram (d), the tensile fracture strain of the orthogonal collagen fiber sandwich structure in experimental group b reached 145.1%, the tensile fracture strain of the unidirectional collagen fiber sandwich structure in experimental group a reached 179.6%, and the tensile fracture strain of the fiber-free structure in the blank control group reached 129.7%. The tensile fracture strain of the random collagen fiber sandwich structure in experimental group c, while lower than that in experimental group a, was still higher than that in the blank control group. This shows that the collagen fiber sandwich structure exhibits excellent tensile fracture strain performance, with the unidirectional collagen fiber sandwich structure achieving the highest tensile fracture strain.

[0097] like Figure 6 As shown, the swelling performance test of the artificial corneas of experimental group a and experimental group b showed that the orthogonal collagen fiber sandwich structure of experimental group b and the unidirectional collagen fiber sandwich structure of experimental group a had low swelling properties, while the fiber layer-free structure of the blank control group showed obvious swelling.

[0098] like Figure 7 As shown, the transmittance of the artificial corneas of experimental group a and experimental group b was tested. Compared with the natural cornea, it can be seen that the orthogonal collagen fiber sandwich structure of experimental group b and the unidirectional collagen fiber sandwich structure of experimental group a both have good transmittance in the visible light band.

[0099] like Figures 8 to 10 As shown, the biocompatibility and cell behavior of the artificial corneas were verified. In the CCK8 toxicity test, artificial corneas with different orthogonal collagen fiber sandwich structures were prepared (experimental group I: 6 layers; experimental group II: 10 layers; control group I: 0 layers). These artificial corneas were immersed in DMEM / F12 cell culture medium for 24 hours to obtain artificial corneal extracts. The artificial corneal extracts were incubated with human corneal fibroblasts for 6, 12, and 24 hours, and cell viability exceeded 95%, with significant proliferation.

[0100] Cell survival and skeletal expression, the bio-ink was heated to 37 ° C for liquefaction, scraped to form a thickness of 50 μm, and cooled to 18 ° C for gelation; the collagen fiber membrane was attached to the gel layer in the X-axis direction, and DLP projection (light intensity 25mW / cm², exposure 15s) was used to solidify the current layer to obtain an experimental group with an oriented collagen fiber membrane on the surface. The bio-ink was heated to 37 ° C for liquefaction, scraped to form a thickness of 50 μm, and cooled to 18 ° C for gelation, and DLP projection (light intensity 25mW / cm², exposure 15s) was used to solidify the current layer to obtain a control group with only bio-ink gel. Figure 9 As shown in the live-death staining, the cell viability on the ordered collagen fiber membrane surface and the gel surface was >90%. Figure 10 As shown in the cytoskeleton staining, F-actin fluorescence showed that the cytoskeleton of the experimental group was highly ordered and consistent with the fiber direction.

[0101] It can be seen that the present invention uses photofunctionalized modified collagen fibers and GelMA / HA-DN, and accurately coordinates the alternating stacking process of oriented arranged collagen fiber membranes obtained through phase change DLP 3D printing technology and electrospinning technology to achieve efficient molding of artificial corneas that imitate natural orthogonal collagen fiber sandwich structures.

[0102] Specifically, (1) the present invention achieves precise realization of biomimetic structure: compared with the conventional 3D printing (such as extrusion molding) in the prior art, which can only realize the continuous stacking of a single material, and cannot embed directional fibers or form an orthogonal arrangement between layers (such as the single fiber layer of the CN119139549A patent); or the molding method, which requires step-by-step assembly of the fiber membrane and the matrix material, the interlayer bonding force is weak and easy to dislocate. The present invention, however, uses phase change DLP 3D printing and electrospinning technology to prepare an ordered photoresponsive collagen fiber membrane, which achieves seamless chemical bonding between layers through in-situ photocrosslinking, thereby improving the interface strength. (2) The present invention achieves high-precision control of ultra-thin layers: utilizing the temperature-sensitive properties of GelMA, the rapid conversion between liquid layering and gelation molding is achieved through temperature switching, ensuring the uniformity of the single layer thickness. The layer thickness of the present invention is close to that of the natural corneal stroma (about 2μm single layer thickness), and the multi-layer orthogonal stacking simulates the natural mechanical gradient. (3) The present invention achieves dynamic cross-linking synergy and enhances structural stability: Compared with the existing UV single cross-linking method that relies only on chemical cross-linking, the fibers are extremely prone to shrinkage and deformation during curing (shrinkage rate > 50%); while pure physical cross-linked scaffolds (such as cryogels) are prone to swelling and decomposition in the body fluid environment. The present invention first temporarily fixes the scaffold through physical cross-linking, effectively reducing the shrinkage rate (<= 3%), and then the chemical cross-linking network effectively reduces the swelling rate to below 10%. (4) The present invention achieves multi-material integration: rigid collagen fiber membranes and flexible GelMA / HA-DN hydrogels are alternately stacked to simulate the microenvironment of the natural cornea, increase functional gradient design, use high-density ordered fibers to enhance suture strength, and the interlayer porous hydrogel promotes cell migration. This rigid-flexible composite structure can reduce the rejection reaction caused by interface stress concentration, support directional cell movement, and accelerate postoperative healing.

[0103] In summary, the present invention achieves the coordinated optimization of ultra-low swelling, high transmittance, bionic mechanics and biocompatibility in the manufacture of artificial corneas through a closed-loop design of material modification-structural bionics-process innovation. It breaks through the contradiction of "high transmittance must sacrifice mechanics" and "low swelling requires reliance on synthetic materials" in existing strategies, and provides a solution for ophthalmic implants that is closer to the performance of natural corneas. It can also be expanded to other biomedical devices with bionic layered structures (such as heart valves, cartilage, skin scaffolds, etc.).

[0104] The embodiments of the present invention are described in detail above, but the contents are only preferred embodiments of the present invention and should not be considered to limit the scope of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the scope of the present invention.

Claims

1. A method for preparing an ultra-low swelling artificial cornea that mimics the fiber arrangement of a natural cornea, characterized by: The following steps are involved: S1. Preparation of photofunctionalized collagen: Dissolve type I collagen in phosphate buffer, add active reagent for photofunctionalization modification for chemical modification, stir in the dark, and freeze-dry to obtain photofunctionalized collagen; S2. Preparation of collagen fiber membrane: dissolving the photofunctionalized collagen from step S1, and then preparing collagen fiber membrane by electrospinning; S3, phase change DLP 3D printing to construct collagen fiber sandwich structure; S31. Preparing a bio-ink for phase change DLP 3D printing: mixing methacrylated gelatin, HA-DN, and a photoinitiator to form a temperature-sensitive liquid bio-ink; S32, constructing a collagen fiber sandwich structure layer by layer: using the bio-ink prepared in step S31 and the collagen fiber membrane prepared in step S2 to perform phase change DLP 3D bioprinting to obtain a collagen fiber sandwich structure; S4, returning the collagen fiber sandwich structure formed in step S3 to room temperature, removing the uncrosslinked bio-ink with a phosphate solution, and obtaining an artificial cornea with a stable collagen fiber sandwich structure; The number of layers of the collagen fiber sandwich structure of the artificial cornea is 3 to 20; the swelling rate of the artificial cornea is not higher than 10%, and the light transmittance is greater than 85%; In step S1, the concentration of the phosphate buffer is 0.5% to 4%; the amount of the active agent for photofunctionalization modification is 2% to 10% of the mass of type I collagen, and the active agent for photofunctionalization modification is methacrylic anhydride or N-hydroxysuccinimide acrylate containing a C=C functional group; In step S2, the collagen fiber membrane includes an ordered collagen fiber membrane and a random collagen fiber membrane; in step S3, the collagen fiber sandwich structure includes an orthogonal collagen fiber sandwich structure, a unidirectional collagen fiber sandwich structure and a random collagen fiber sandwich structure; In step S31, the concentration of the methacrylated gelatin is 5% to 15%, the HA-DN is formed by mixing aldehyde-modified hyaluronic acid and hydrazide-modified hyaluronic acid to form a dynamic covalent bond, and the mass ratio of the aldehyde-modified hyaluronic acid to the hydrazide-modified hyaluronic acid is 1:1 to 1:3; the photoinitiator is LAP, and the concentration of the LAP is 0.1% to 0.5%; the LAP is a 405 nm wavelength-sensitive initiator; In step S32, the printing process includes the following steps: S321, preheating the bio-ink: heating the bio-ink to 37-45°C, and coating the bio-ink on the printing platform with a scraper to form a uniform ultra-thin layer with a thickness of 10-50 μm; S322, low temperature gelation: cooling to below 25°C to obtain a gel layer; S323, collagen fiber membrane laying: attaching the collagen fiber membrane to the surface of the gel layer prepared in step S322 to achieve close adhesion; S324, In-situ patterned photocrosslinking: Using the DMD in the DLP 3D printing system to perform patterned projection and solidification of the current layer, chemically crosslinking the collagen fiber membrane and the bio-ink layer; S325, alternating stacking: repeat steps S321 to S324 to arrange the next layer of collagen fiber membrane, and finally form a multi-layer collagen fiber sandwich structure.

2. The method for preparing an ultra-low swelling artificial cornea with natural corneal fiber arrangement according to claim 1, characterized in that: In step S1, stirring is performed at room temperature (25-37° C.) in the dark for 6-12 hours; and then freeze-drying is performed at -50--80° C. and a vacuum degree of less than or equal to 1 Pa for 18 hours.

3. The method for preparing an ultra-low swelling artificial cornea with natural corneal fiber arrangement according to claim 1, characterized in that: In step S2, the photofunctionalized collagen is dissolved in hexafluoroisopropanol or an acetic acid / water mixed solvent.

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