Ultralow-swelling artificial cornea imitating natural cornea fiber arrangement and preparation method of ultralow-swelling artificial cornea

Abstract

The invention provides an ultralow-swelling artificial cornea imitating natural cornea fiber arrangement and a preparation method, and belongs to the technical field of biomedical materials.The preparation method comprises the steps that I-type collagen is dissolved in a PBS buffer solution, a photofunctionalization modified active reagent is added for chemical modification, stirring is conducted in a dark place, freeze drying is conducted, and photofunctionalization collagen is obtained; dissolving the photo-functionalized collagen, and then preparing a collagen fiber membrane through electrostatic spinning; and performing phase change DLP 3D biological printing on the biological ink and the collagenous fiber membrane to obtain the keratoprosthesis with the collagenous fiber sandwich structure. Multi-layer arrangement is achieved through DLP 3D biological printing, the fiber trend of the natural cornea is simulated, and the mechanical anisotropy is remarkably improved; through a composite phase change DLP 3D printing mode of GelMA / HA-DN and photo-crosslinking collagenous fibers, the composite phase change DLP 3D printing material has good manufacturability, maintains high light transmittance and maintains high mechanical strength.

Description

Technical Field

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

[0002] An artificial cornea refers to a special refractive device made of artificial synthetic materials, which is used to replace the cloudy cornea that hinders the optical path of the eyeball after the disease, so that the patient can obtain a certain vision.

[0003] Current mainstream artificial cornea materials include high molecular polymers (such as the Boston type artificial cornea of polymethyl methacrylate PMMA / PHEMA), hydrogels (such as polyethylene glycol derivatives), and biological materials (such as acellular corneal matrix). Among them, synthetic high molecular polymer materials and hydrogel materials have stable and controllable mechanical properties, but poor biocompatibility, resulting in serious wear or corneal ablation in the later stage, and their lack of bionic fiber arrangement makes it impossible to integrate with host tissues. Natural biological materials are similar to natural tissues in composition and have good biocompatibility, but poor mechanical properties, and there are problems such as complex processes, difficult restoration of light transmittance, and the risk of immune rejection. In addition, electrospinning technology can be used to prepare collagen fiber membranes, but the fiber arrangement of collagen fiber membranes is mostly disordered or unidirectional, lacking bionic orthogonal structure design.

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

[0005] At present, the application of tissue engineering in the preparation of artificial cornea has important scientific significance and clinical value. Its core lies in breaking through the limitations of traditional artificial corneas through the combination of bionic design, biomaterials and cell technology, and providing innovative solutions to solve the problems of corneal donor shortage, postoperative complications and functional recovery. In the prior art, tissue engineering corneal scaffolds mostly use decellularized matrix or collagen scaffold combined with cell culture, such as patent CN119139549A, which assembles collagen fiber membranes through electric field triggered dynamics, but only relies on a single riboflavin cross-linked natural collagen component, and does not introduce other high bond energy synergistic cross-linking. This method is difficult to control the swelling rate and mechanical properties at the same time, resulting in the material being easily degraded or deformed in the body fluid environment, and the cross-linking stability is poor. At the same time, the existing strategy 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 that the use of intelligent manufacturing means to prepare orthogonal grids can obtain artificial corneas with certain collagen fiber microstructures, but the cross-linking method of the structure is uncertain, and the 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 imitates the fiber arrangement of the natural cornea and a preparation method. Through the coordinated design of collagen photocross-linking modification and multi-layer collagen fiber sandwich structure based on phase change DLP 3D bio-printing, combined with the physical phase change characteristics of light curing technology and thermosensitive materials, the efficient and high-precision preparation of complex structures is achieved by dynamically regulating the cross-linking method (cooperation of photochemical cross-linking and physical cross-linking), which solves the technical problems in the prior art of low mechanical strength and decreased transmittance of the artificial cornea caused by high material swelling rate, insufficient bionics of fiber arrangement and poor cross-linking stability.

[0007] In order to solve the above technical problems, the technical solution adopted by the present invention is: a method for preparing an ultra-low swelling artificial cornea imitating 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 reagents 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 for photofunctional modification is 2% to 10% of the mass of type I collagen, and the active agent for photofunctional modification is methacrylic anhydride (MA) or N-hydroxysuccinimide acrylate (NHS-acrylate) containing a C=C functional group.

[0011] Further, in step S1, under room temperature conditions of 25~37°C, stir in the dark for 6~12 h; then freeze-dry for 18 h under the conditions of -50~-80°C and a vacuum degree of less than or equal to 1 Pa.

[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 bioink during subsequent DLP printing, inhibiting swelling, and enhancing interfacial bonding strength.

[0013] S2. Preparation of collagen fiber membrane: Dissolve the photo-functionalized collagen in step S1, and then prepare a collagen fiber membrane by electrospinning.

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

[0015] Further, in step S2, the photo-functionalized 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] Further, in step S2, the parameters of the electrospinning are: voltage 15~40 kV, spinning solution flow rate 0.5~2 ml / h, receiving distance 10~30 cm, and received by a rotating receiving device with a rotation speed of 500~5000 rpm.

[0017] S3. Construct a collagen fiber sandwich structure by phase change DLP 3D printing.

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

[0019] Further, in step S31, the concentration of GelMA is 5%~15% (w / v). GelMA is a temperature-sensitive phase change material that realizes temporary physical crosslinking support through temperature regulation (gelation at 25°C and liquid state at 37°C). HA-DN is formed by mixing aldehyde group-modified hyaluronic acid (HA-CHO) and hydrazide group-modified hyaluronic acid (HA-ADH). The mass ratio of HA-CHO to HA-ADH is 1:1~1:3. The hyaluronic acid is respectively modified with an aldehyde group (-CHO) and a hydrazide group (-ADH) to form a dynamic covalent bond (Schiff base bond), endowing the material with tear resistance and self-healing ability. The concentration of the photoinitiator LAP is 0.1%~0.5%. LAP is a 405 nm wavelength-sensitive initiator that reduces the damage of ultraviolet light to biological activity.

[0020] S32, constructing a collagen fiber sandwich structure layer by layer: using the biological 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.

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

[0022] S321, preheating the bio-ink: heating the bio-ink to 37-45°C to ensure good fluidity, and coating the bio-ink on the printing platform with a scraper in conjunction with a high-precision displacement system 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, laying collagen fiber membrane: attaching the collagen fiber membrane to the surface of the gel layer obtained in step S322, and fixing the position of the collagen fiber membrane by surface tension to achieve close fitting;

[0025] S324, In-situ patterned photo-crosslinking: Use the DMD in the DLP 3D printing system for patterned 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 biological 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 biological ink form a chemical cross-linking network to increase the interface strength;

[0026] S325, alternate 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 rewarmed to 37°C and maintained for 5-10 minutes. The bio-ink (GelMA / HA) in the area not projected with light 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 layers.

[0031] Furthermore, the swelling rate of the artificial cornea is not higher than 10%, and the light transmittance is greater than 85%.

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

[0033] The present invention provides a low-swelling artificial cornea with a collagen fiber sandwich structure and a preparation method thereof. Through the collaborative innovation of photo-functionalized modified collagen fibers, electrospinning technology and phase-change DLP 3D bioprinting process, it solves the problems of decreased light transmittance and poor biocompatibility caused by high swelling rate, insufficient mechanical strength and lack of bionic structure in the existing artificial corneas. It can be further extended to other bionic layered structure biomedical devices, such as heart valves, cartilage, skin scaffolds, etc.

[0034] (1) After the collagen fibers of the present invention are photo-functionalized and modified, they are arranged in multiple layers through DLP 3D bioprinting, simulating the fiber orientation of the natural cornea, and significantly improving the mechanical anisotropy (the tensile strength is improved). The composite phase-change DLP 3D printing method of GelMA / HA-DN and photo-crosslinked collagen fibers makes it have good manufacturability, maintains a high light transmittance (>90%), and at the same time maintains high mechanical strength.

[0035] (2) The bionic microstructure and surgical adaptability of the present invention: The sandwich design (fiber membrane-hydrogel-fiber membrane) enhances the suture stability, inhibits the swelling rate (ultra-low swelling), and reduces the risk of postoperative graft displacement. Among them, the orthogonally arranged fiber network promotes cell directional migration, accelerates the integration with the host cornea, and reduces the rejection reaction. Description of the Drawings

[0036] The present invention will be specifically described below with reference to the drawings and in combination with examples. The advantages and implementation methods of the present invention will become more obvious. The content shown in the drawings is only used for the explanation of the present invention and does not constitute any limitation to the present invention. In the drawings:

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

[0038] Figure 2 It is the contact angle test image of the collagen fiber membrane of the present invention.

[0039] Figure 3 It is the rheological test image of the bioink of the present invention.

[0040] Figure 4 It is the physical image of the self-healing performance of the bioink of the present invention.

[0041] Figure 5 It is the mechanical test image of the artificial cornea of the present invention.

[0042] Figure 6 This is the swelling rate graph of the artificial cornea of the present invention.

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

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

[0045] Figure 9 This is the graph of the viability of cells on the surface of the artificial cornea of the present invention.

[0046] Figure 10 This is the graph of the cytoskeleton expression of the artificial cornea of the present invention. Detailed implementation manners

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

[0048] A preparation method of an ultra-low swelling artificial cornea imitating the fiber arrangement of natural corneas includes the following steps:

[0049] S1. Preparation of photo-functionalized collagen: Dissolve type I collagen in a phosphate buffer solution (PBS) with a concentration of 0.5% - 4%, add methacrylic anhydride (MA) or N-hydroxysuccinimide acrylate (NHS-acrylate) containing a C=C functional group as an active reagent for photo-functionalization modification for chemical modification, and stir in the dark for 6 - 12 h at room temperature (25 - 37°C); then freeze-dry for 18 h under the conditions of -50 to -80°C and a vacuum degree less than or equal to 1 Pa to obtain photo-functionalized collagen (photo-responsive collagen);

[0050] Among them, the addition amount of the active reagent for photo-functionalization modification is 2% - 10% of the mass of type I collagen.

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

[0052] S2. Preparation of collagen fiber membrane: Dissolve the photo-functionalized collagen obtained in step S1 in hexafluoroisopropanol (HFIP) or an acetic acid / water mixed solvent (the volume ratio of acetic acid to water is 8:2), and then prepare a collagen fiber membrane through electrospinning technology.

[0053] Among them, the collagen fiber membrane includes an ordered collagen fiber membrane and a random collagen fiber membrane.

[0054] The electrospinning parameters for the preparation of 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 membrane (fiber diameter 50~500 nm) was prepared by directional spinning technology to simulate the natural ordered fiber arrangement.

[0056] The electrospinning parameters for preparing random collagen fiber membranes were: voltage 15-40 kV, spinning solution flow rate 0.5-2 ml / h, receiving distance 25 cm, and received by a rotating receiving device (slow-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 structure.

[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] Among them, the concentration of GelMA is 5%~15% (w / v). GelMA is a thermosensitive phase change material that achieves temporary physical cross-linking support 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). The mass ratio of HA-CHO to HA-ADH is 1:1~1:3. Hyaluronic acid is modified with aldehyde (-CHO) and hydrazide (-ADH) to form dynamic covalent bonds (Schiff base bonds), which give the material tear resistance and self-healing ability. The concentration of the photoinitiator LAP is 0.1%~0.5%: LAP is a 405 nm wavelength-sensitive initiator that reduces the damage of ultraviolet light to biological activity.

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

[0061] S321, preheating of bio-ink: heat the bio-ink to 37~45℃ to ensure good fluidity, and 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: Cool down to below 25 °C (preferably 15 - 20 °C). GelMA rapidly physically cross-links through hydrogen bonds to form a stable support, achieving physical gelation and obtaining a gel layer;

[0063] S323. Collagen fiber membrane layering: Attach the collagen fiber membrane to the surface of the gel layer prepared in step S322, and utilize surface tension to fix the position of the collagen fiber membrane to achieve tight fitting;

[0064] S324. In-situ patterned photocrosslinking: Use the DMD in the DLP 3D printing system for patterned projection (parameters: wavelength 405 nm, light intensity 10 - 50 mW / cm 2 , exposure time 5 - 30 s) to cure the current layer, enabling the collagen fiber membrane and the bioink layer to form a chemical crosslink, that is, the photo-functionalized collagen fibers of the collagen fiber membrane and the GelMA / HA-DN layer of the bioink form a chemical crosslinking network to increase the interfacial strength;

[0065] S325. Alternating stacking:

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

[0067] Unidirectional collagen fiber sandwich structure: Use ordered collagen fiber membranes, repeat steps S321 to S324, and the next layer of ordered collagen fiber membrane is arranged in the same direction as the attachment direction in step S323 (for example, if the attachment direction in step S323 is the X-axis direction, then the attachment direction of the next layer of ordered collagen fiber membrane is also the X-axis direction), finally forming a multi-layer unidirectional collagen fiber sandwich structure (total number of layers 3 - 20 layers);

[0068] Random collagen fiber sandwich structure: Use random collagen fiber membranes, repeat steps S321 to S324, and the next layer of random collagen fiber membrane is randomly attached, finally forming a multi-layer random collagen fiber sandwich structure (total number of layers 3 - 20 layers);

[0069] S4. Rewarm the collagen fiber sandwich structure formed in step S3 to 37 °C and maintain for 5 - 10 min. The bioink (GelMA / HA) in the area not exposed to light projection returns to the liquid state, and the uncrosslinked bioink is gently rinsed off with PBS solution to obtain an artificial cornea with a stable collagen fiber sandwich structure.

[0070] The collagen fiber sandwich structure prepared by the construction method of the present invention can effectively simulate the mechanical anisotropy of the natural cornea and improve the overall tensile strength. At the same time, due to the increased interfacial interaction, the penetration of water molecules can be restricted, and combined with the hydrophobic effect of the HA dynamic bond, the swelling rate can be reduced to less than 10%. In addition, the ordered fiber arrangement and the method of photocrosslinking can effectively fix the fiber layer while reducing light scattering and optimizing the light transmittance of the artificial cornea.

[0071] Through the full-process innovation of photo-functionalization modification - electrospinning orientation - phase change DLP printing, the present invention realizes the synergistic optimization of ultra-low swelling, high light transmittance and bionic mechanical properties of the artificial cornea, filling the gap that it is difficult to balance material properties and structural bionics in traditional technologies.

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

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

[0074] The swelling rate of the artificial cornea is not higher than 10%, and the light transmittance is greater than 85%.

[0075] Example:

[0076] A preparation method of an ultra-low swelling artificial cornea with a fiber arrangement imitating that of the natural cornea includes the following steps:

[0077] S1. Preparation of photo-functionalized collagen: Dissolve type I collagen in PBS buffer solution with a concentration of 2%, add N-hydroxysuccinimide acrylate with a mass of 10% of type I collagen for chemical modification, and stir in the dark at room temperature for 6 h; then freeze-dry in a freeze dryer at -70°C and a vacuum degree of 0.8 Pa for 18 h to obtain white porous sponge-like photo-functionalized collagen (photo-responsive collagen);

[0078] S2. Preparation of an ordered collagen fiber membrane: Dissolve the photo-functionalized collagen in step S1 in HFIP with a concentration of 8%, load it into a 20 ml syringe, set the parameters of electrospinning as: voltage 20 kV, spinning solution flow rate 1 ml / h, receiving distance 15 cm, and receive through a high-speed rotating drum with a rotation speed of 3000 rpm, and the spinning lasts for 2 h. Figure 1 As shown, an ordered collagen fiber membrane with a fiber diameter of about 200 nm and a unidirectional arrangement is obtained.

[0079] As Figure 2As shown, the contact angle test was carried out, and the contact angle of the prepared collagen fiber membrane was 35° (the bioink layer was 60°), indicating that the cross-linked collagen fibers are more hydrophilic and beneficial to cell adhesion.

[0080] Preparation of random collagen fiber membrane: Dissolve the photo-functionalized collagen in step S1 in HFIP with a concentration of 8%, load it into a 20 ml syringe, and set the parameters of electrospinning as follows: voltage 20 kV, spinning solution flow rate 1 ml / h, receiving distance 25 cm, and receive it through a low-speed rotating drum with a rotation speed of 500 rpm. The spinning lasts for 2 h.

[0081] S3. Phase change DLP 3D printing: Construct a collagen fiber sandwich structure by phase change DLP 3D printing.

[0082] S31. Preparation of bioink 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, and stir at 37°C for 2 h until completely dissolved to form a temperature-sensitive liquid bioink.

[0083] As Figure 3 and Figure 4 shown, Figure 3 (a) is the frequency conversion test chart. Frequency scanning (0.1~10 Hz) shows that the storage modulus (G') of the bioink with HA-DN added (experimental group 1 and experimental group 2, where the concentration of HA-DN in experimental group 2 is higher than that in experimental group 1) is significantly improved (the control group is pure GelMA). At the same time, as the concentration of HA-DN further increases, the increase in the storage modulus is limited. Figure 3 (b) is the photocuring test chart. Under 405 nm light illumination, the complex viscosity of the bioink is significantly enhanced within 30 s, proving the photo-controlled polymerization ability. After photocuring, the complex viscosity increases with the increase in the concentration of HA-DN. Combining the results of the frequency conversion experiment, the preferred concentration of HA-DN is 0.3%, and this concentration is also used in subsequent experiments.

[0084] As Figure 4 shown, the cut bioink gel can heal together and have a certain tear resistance after standing at room temperature for 30 min, indicating that the used bioink layer forms dynamic covalent bonds and has good self-healing performance.

[0085] S32. Layer-by-layer construction of the collagen fiber sandwich structure: Use the bioink prepared in step S31 and the collagen fiber membrane prepared in step S2 for phase change DLP 3D bioprinting. The printing process includes the following steps:

[0086] S321, preheating of bio-ink: heat the bio-ink to 37°C to ensure good fluidity, and use a scraper with a high-precision displacement system to coat the printing platform to form a uniform ultra-thin layer with a thickness of 50 μm;

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

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

[0089] S324, in-situ patterned photo-crosslinking: using the DMD in the DLP 3D printing system for patterned 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 biological 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 biological ink form a chemical cross-linking network to increase the interface strength;

[0090] S325, alternate stacking:

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

[0092] Experimental group b: orthogonal collagen fiber sandwich structure: using ordered collagen fiber membrane, repeating steps S321 to S324, the next layer of ordered collagen fiber membrane is arranged in the orthogonal direction of the attachment direction in step S323, and finally forming 10 layers of orthogonal collagen fiber sandwich structure hydrogel (total thickness of about 500 μm);

[0093] Experimental group c: random collagen fiber sandwich structure: using random collagen fiber membrane, repeating steps S321 to S324, and randomly attaching the next layer of random collagen fiber membrane, finally forming 10 layers of 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 groups a, b, and the blank control group formed in step S3 to 37 °C and maintain for 5 - 10 min. The bioink (GelMA / HA) in the non-light-projected area returns to the liquid state, and the uncrosslinked bioink is gently rinsed off with PBS solution to obtain artificial corneas respectively.

[0096] As Figure 5 shown, for the mechanical properties of compression and stretching of the above artificial corneas, Figure 5 In the compression property test diagram of (a), the maximum compression modulus of the orthogonal collagen fiber sandwich structure in experimental group b reaches 693.3 kPa, the maximum compression modulus of the unidirectional collagen fiber sandwich structure in experimental group a is 633.5 kPa, and the maximum compression modulus of the fiber-free layer structure in the blank control group is 120.7 kPa; although the maximum compression modulus of the random collagen fiber sandwich structure in experimental group c is lower than that in experimental groups a and b, it is also much higher than that in the blank control group. It can be seen that the collagen fiber sandwich structure has excellent compression properties, among which the orthogonal collagen fiber sandwich structure has the best compression performance. Figure 5 In the compression property test diagram of (b), the compression fracture strain of the orthogonal collagen fiber sandwich structure in experimental group b reaches 94.5%, the compression fracture strain of the unidirectional collagen fiber sandwich structure in experimental group a is 79%, and the compression fracture strain of the fiber-free layer structure in the blank control group is 60.5%; although the compression fracture strain of the random collagen fiber sandwich structure in experimental group c is lower than that in experimental group b, it is also higher than that in the blank control group. It can be seen that the collagen fiber sandwich structure has excellent compression fracture strain properties, among which the orthogonal collagen fiber sandwich structure has the best compression fracture strain performance. Figure 5 In the tensile property test diagram of (c), the maximum tensile modulus of the orthogonal collagen fiber sandwich structure in experimental group b reaches 656.2 kPa, the maximum tensile modulus of the unidirectional collagen fiber sandwich structure in experimental group a is 452.8 kPa, and the maximum tensile modulus of the fiber-free layer structure in the blank control group is 94.05 kPa. Although the maximum tensile modulus of the random collagen fiber sandwich structure in experimental group c is lower than that in experimental groups a and b, it is also much higher than that in the blank control group. It can be seen that the collagen fiber sandwich structure has excellent tensile properties, among which the orthogonal collagen fiber sandwich structure has the best tensile performance. Figure 5 In the tensile property test diagram of (d), the tensile fracture strain of the orthogonal collagen fiber sandwich structure in experimental group b reaches 145.1%, the tensile fracture strain of the unidirectional collagen fiber sandwich structure in experimental group a is 179.6%, and the tensile fracture strain of the fiber-free layer structure in the blank control group is 129.7%; although the tensile fracture strain of the random collagen fiber sandwich structure in experimental group c is lower than that in experimental group a, it is also higher than that in the blank control group. It can be seen that the collagen fiber sandwich structure has excellent tensile fracture strain properties, among which the unidirectional collagen fiber sandwich structure has the best tensile fracture strain.

[0097] As Figure 6 shown, the swelling properties of the artificial corneas of experimental group a and experimental group b were tested. 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 have low swelling properties, while obvious swelling occurred in the fiber-free layer structure of the blank control group.

[0098] As Figure 7 shown, the light transmittance of the artificial corneas of experimental group a and experimental group b was tested. Comparing 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 light transmittance in the visible light band.

[0099] As Figures 8 to 10 shown, the biocompatibility and cell behavior of the above 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), and they were immersed in DMEM / F12 cell culture medium for 24 h to obtain artificial cornea extracts. The artificial cornea extracts were cultured with human corneal fibroblasts for 6, 12, and 24 h, and the cell viability > 95%, and there was obvious proliferation.

[0100] Cell survival and cytoskeleton expression. The bioink was heated to 37 °C to liquefy, scraped and coated to form a thickness of 50 μm, and cooled to 18 °C to gelify; the collagen fiber membrane was attached to the gel layer in the X-axis direction, and the current layer was cured by DLP projection (light intensity 25 mW / cm², exposure 15 s) to obtain an experimental group with an oriented collagen fiber membrane on the surface. The bioink was heated to 37 °C to liquefy, scraped and coated to form a thickness of 50 μm, and cooled to 18 °C to gelify, and the current layer was directly cured by DLP projection (light intensity 25 mW / cm², exposure 15 s) to obtain a control group with only bioink gel. As Figure 9 shown, in the live-dead staining, the cell survival rates on the surface of the ordered collagen fiber membrane and the gel surface were both > 90%. As Figure 10 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 photo-functionalized modified collagen fibers and GelMA / HA-DN, and through the precise cooperation of the alternating stacking process of the phase change DLP 3D printing technology and the electrospinning technology to obtain an oriented arrangement of collagen fiber membranes, realizing the efficient forming of the artificial cornea imitating the natural orthogonal collagen fiber sandwich structure.

[0102] Specifically, (1) the present invention achieves precise realization of bionic structure: compared with the conventional 3D printing (such as extrusion molding) in the prior art, it can only realize the continuous stacking of a single material, and cannot embed oriented fibers or form an orthogonal arrangement between layers (such as the single fiber layer of the CN119139549A patent); or in the molding method, the fiber membrane and the matrix material need to be assembled step by step, and the interlayer bonding force is weak and easy to dislocate. However, the present invention uses phase change DLP 3D printing and electrospinning technology to prepare an ordered photoresponsive collagen fiber membrane, which realizes seamless chemical bonding between layers through in-situ photocrosslinking to improve the interface strength. (2) The present invention realizes high-precision control of ultra-thin layers: utilizing the temperature-sensitive properties of GelMA, the rapid conversion between liquid layering and gelation molding is realized through temperature switching to ensure the uniformity of single-layer thickness. The layer thickness of the present invention is close to the natural corneal stroma layer (about 2μm single-layer thickness), and the multi-layer orthogonal stacking simulates the natural mechanical gradient. (3) The present invention realizes dynamic cross-linking synergy and enhances structural stability: Compared with the existing UV single cross-linking method that only relies on chemical cross-linking, the fibers are very easy to shrink and deform during curing (shrinkage rate>50%); while pure physical cross-linked scaffolds (such as cryogel) are easy to swell and decompose in the body fluid environment. The present invention first temporarily fixes by physical cross-linking to effectively reduce the shrinkage rate (<=3%), and then the chemical cross-linking network effectively reduces the swelling rate to below 10%. (4) The present invention realizes 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 the functional gradient design, use high-density ordered fibers, 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, breaking through the contradiction of "high transmittance must sacrifice mechanics" and "low swelling requires reliance on synthetic materials" in existing strategies, providing a solution for ophthalmic implants that is closer to the performance of natural corneas, and can be expanded to other bionic layered biomedical devices (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 cannot be considered to limit the scope of implementation 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 natural corneas, characterized by: It includes the following steps: S1. Preparation of photo-functionalized collagen: Dissolve type I collagen in PBS buffer, add a photo-functionalized active reagent for chemical modification, stir in the dark, and then freeze-dry to obtain photo-functionalized collagen. S2. Preparation of collagen fiber membrane: Dissolve the photo-functionalized collagen obtained in step S1, and then prepare a collagen fiber membrane by electrospinning. S3. Construct a collagen fiber sandwich structure by phase-change DLP 3D printing. S31. Prepare the bio-ink for phase-change DLP 3D printing: Mix GelMA, HA-DN, and a photo-initiator to form a temperature-sensitive liquid bio-ink. S32. Layer-by-layer construct the collagen fiber sandwich structure: Use the bio-ink prepared in step S31 and the collagen fiber membrane prepared in step S2 for phase-change DLP 3D bioprinting to obtain a collagen fiber sandwich structure. S4. Rewarm the collagen fiber sandwich structure formed in step S3 to room temperature, and remove the uncrosslinked bio-ink through PBS solution to obtain an artificial cornea with a stable collagen fiber sandwich structure.

2. The preparation method of the ultra-low swelling artificial cornea imitating the fiber arrangement of the natural cornea according to claim 1, characterized in that: In step S1, the concentration of the PBS buffer is 0.5% - 4%; the addition amount of the photo-functionalized active reagent is 2% - 10% of the mass of type I collagen, and the photo-functionalized active reagent is methacrylic anhydride or N-hydroxysuccinimide acrylate containing a C=C functional group.

3. The preparation method of the ultra-low swelling artificial cornea imitating the fiber arrangement of natural cornea according to claim 1, wherein: In step S1, under the conditions of room temperature (25 - 37 °C), stir in the dark for 6 - 12 h; then freeze-dry at -50 to -80 °C under a vacuum of less than or equal to 1 Pa for 18 h.

4. The preparation method of the ultra-low swelling artificial cornea imitating the fiber arrangement of natural cornea according to claim 1, characterized in that: In step S2, the photo-functionalized collagen is dissolved in hexafluoroisopropanol or an acetic acid / water mixed solvent.

5. The preparation method of the ultra-low swelling artificial cornea imitating the fiber arrangement of natural cornea according to claim 1, characterized in that: In step S31, the concentration of GelMA is 5% - 15%, HA-DN is formed by mixing HA-CHO and HA-ADH to form a dynamic covalent bond, and the mass ratio of HA-CHO to HA-ADH is 1:1 - 1:3; the photo-initiator is LAP, and the concentration of LAP is 0.1% - 0.5%.

6. The preparation method of the ultra-low swelling artificial cornea with fiber arrangement imitating natural cornea according to claim 1, characterized in that: Further, in step S32, the printing process includes the following steps: S321. Preheat the bio-ink: Heat the bio-ink to 37 - 45 °C, and coat it on the printing platform with a spatula to form a uniform ultra-thin layer, and the thickness of the ultra-thin layer is 10 - 50 μm. S322. Low-temperature gelation: Cool down to below 25 °C to obtain a gel layer. S323. Lay the collagen fiber membrane: Attach the collagen fiber membrane to the surface of the gel layer prepared in step S322 to achieve a tight fit. S324. In-situ patterned photo-crosslinking: Use the DMD in the DLP 3D printing system to perform patterned projection curing on the current layer to form a chemical crosslink between the collagen fiber membrane and the bio-ink layer. S325. Alternate 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.

7. The preparation method of the ultra-low swelling artificial cornea imitating the fiber arrangement of natural cornea according to claim 1, characterized in that: 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.

8. An artificial cornea, characterized in that: It is prepared by the method for preparing an ultra-low swelling artificial cornea with a fiber arrangement imitating natural cornea according to any one of claims 1 to 7.

9. The artificial cornea according to claim 8, wherein: The number of layers of the collagen fiber sandwich structure of the artificial cornea is 3 to 20 layers.

10. The artificial cornea according to claim 8, wherein: The swelling rate of the artificial cornea is not higher than 10%, and the light transmittance is greater than 85%.

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