Spoke-shaped acellular cornea and preparation method and application thereof

Abstract

The application discloses a spoke-shaped decellularized cornea and a preparation method and application thereof, and belongs to the technical field of biomaterials and tissue engineering. The application realizes an orderly process of the phase change of water molecules along the temperature gradient direction by establishing a gradient temperature field from the periphery to the center of a sheet-shaped biological corneal tissue, and forms spoke-shaped microchannels in the cornea. The spoke-shaped microchannels can accelerate the decellularization process of the central region of the cornea, avoid excessive decellularization of the peripheral region of the cornea to cause excessive loss of bioactive components of the extracellular matrix, form a biomimetic migration path between the cornea and the receptor, facilitate the migration and infiltration of the cells of the receptor tissue into the cornea, promote the biological healing of the transplanted cornea and the limbus of the receptor cornea, and improve the biological stability of the transplanted cornea.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials and tissue engineering technology, specifically relating to a spoke-shaped decellularized cornea, its preparation method, and its application. Background Technology

[0002] The cornea is one of the most important tissues for maintaining normal vision. The natural cornea consists of the epithelial cell layer, the anterior elastic lamina, the stroma, the des elastic lamina, and the endothelial cell layer from the outside in. The stroma is a transparent tissue composed of ordered collagen bundles and proteoglycans. The collagen bundles are 22-32 nm in diameter and spaced approximately 30 nm apart, providing the cornea with special biomechanical properties to withstand intraocular pressure. Corneal diseases such as keratoconus, bullous keratosis, fungal infections, and traumatic scarring can cause vision loss or blindness. Currently, corneal blindness is the second leading cause of blindness worldwide.

[0003] Corneal transplantation is the most effective way to restore vision for patients blinded by corneal diseases. Early corneal transplants typically used allogeneic donors, but the number of donated corneas fell far short of the demand. my country performs only about 5,000 corneal transplant surgeries annually, with a donor shortage exceeding 99%. Therefore, the transplantation model relying on donated corneas could not meet clinical needs. Subsequently, scientists began developing various artificial corneas to address the urgent transplantation needs, such as the Boston type, osteodental type, AlphaCor type, and MICOF type. However, these products did not achieve the expected clinical results due to issues such as material biocompatibility or lack of bioactivity.

[0004] In recent years, decellularization technology has made significant progress, and decellularized corneas obtained through this technique are playing an increasingly important role in corneal transplantation. Acellular cornea refers to a biological cornea obtained by removing cellular components from xenogeneic (such as porcine cornea) or allogeneic corneal tissue, reducing immunogenicity and minimizing post-transplant immune rejection, while retaining the extracellular matrix components of the natural cornea. Acellular corneas exhibit good biocompatibility with host tissues, promoting tissue integration, guiding host cell growth and differentiation, and facilitating corneal tissue reconstruction. Furthermore, acellular corneas retain the collagen fiber structure of the natural cornea, possessing excellent mechanical properties, capable of withstanding normal intraocular pressure and external impacts, and their transparency is close to that of the natural cornea, meeting the needs for visual restoration.

[0005] However, the preparation of decellularized corneas using decellularization techniques still faces technical challenges that cannot be addressed by existing technologies, such as excessive decellularization of the periphery while incomplete decellularization occurs in the central region. This is because the sheet-like structure of the cornea and the dense layers on its upper and lower surfaces (anterior and posterior elastic lamina) make the central region a "bottleneck" for decellularization efficiency. Traditional decellularization methods (such as chemical agitation or uniform freeze-thaw) rely on passive diffusion. Removing cell debris from the central cornea requires penetrating the dense upper and lower surfaces, which have significant resistance, or detaching from the periphery through the low-resistance periphery. Achieving overall corneal decellularization requires waiting for sufficient exudation of cellular components from the region near the central cornea. This leads to excessive contact of peripheral corneal tissue with the decellularization agent, resulting in excessive decellularization of the extracellular matrix in the peripheral cornea. Consequently, excessive loss of bioactive components and reduced mechanical properties occur in the peripheral cornea, which is detrimental to maintaining the mechanical stability and bio-inducible properties of subsequent corneal materials.

[0006] To remove as much immunogenic material as possible from the central part of the biological cornea, technicians typically use stronger decellularization agents and extend the decellularization time to ensure effectiveness. This inevitably damages the corneal extracellular matrix and causes loss of bioactive components. Currently, decellularized corneal technology still has significant shortcomings in terms of decellularization efficiency and biocompatibility, necessitating the development of a novel decellularization method to overcome these limitations.

[0007] To address the shortcomings of existing technologies, this invention provides a spoke-shaped decellularized cornea and its preparation method. Before decellularization, the xenograft cornea is cut according to the desired shape for transplantation, and spoke-shaped microchannels are constructed within the cornea. Then, conventional decellularization methods are used for decellularization. This method solves the problem of uneven corneal decellularization in conventional methods and avoids the loss of active components of the extracellular matrix at the corneal limbus. Furthermore, due to the construction of microchannels within the cornea, cells from the recipient corneal limbus can more easily penetrate and migrate, resulting in stronger binding between the transplanted cornea and the recipient, better resistance to the forces exerted on the cornea by intraocular pressure, and a reduced risk of corneal perforation caused by long-term intraocular pressure. Summary of the Invention

[0008] In a specific embodiment of the present invention, a method for preparing a spoke-shaped decellularized cornea is provided. This involves covering the upper and lower surfaces of a biological cornea with a low thermal conductivity insulating material, significantly reducing heat exchange between the cornea and its surfaces in a low-temperature environment. A corneal trephine is used to cut the cornea covered with the insulating material. The cut material, along with the trephine, is placed in a low-temperature environment. Because the trephine is made of stainless steel, its high thermal conductivity allows the peripheral cornea in contact with the trephine to preferentially contact the low-temperature environment, causing ice crystals to preferentially form at the edge of the wet biological cornea. The process of the low-temperature field diffusing from the periphery of the cornea towards the center is controlled in a directional manner, ensuring that the ice crystals always extend towards the warmer central region of the cornea. The ice crystals formed in this process are spoke-shaped crystals with a directional structure, forming spoke-shaped microchannels in the cornea. This method of constructing microchannels in the cornea not only improves the efficiency and extent of subsequent decellularization but also promotes the migration of host tissue cells and facilitates the biointegration of the transplanted cornea with the recipient tissue.

[0009] In the prior art regarding the construction of microchannels in the cornea, patent document CN114904058A discloses a regular porous scaffold. This porous scaffold has lateral microchannels or longitudinal micropores, which are formed by directional freeze-drying, photocuring, or perforation. The directional freeze-drying method involves: placing decellularized matrix material in a directional freeze-drying mold; inserting a metal heat-conducting rod connected to the mold into a cold source; maintaining a fixed temperature within the range of -20 to -196°C, or a programmed cooling or heating process within this temperature range; and after 0.5-4 hours, transferring the mold to a freeze dryer for freeze-drying. The directionally freeze-dried decellularized matrix material forms regularly arranged lateral microchannels or multiple regularly arranged longitudinal micropores. The regular porous scaffold prepared according to this method can be used as a skirt for an artificial cornea. Although this technical solution can form regularly arranged microchannels or micropores inside the decellularized matrix material through the metal heat-conducting rod, the method is not suitable for biological corneas because the metal heat-conducting rod will disrupt the tissue arrangement of the biological cornea itself, causing collagen breakage and damaging the tension of the corneal stroma.

[0010] Those skilled in the art understand that whether the physicochemical properties of the transplanted cornea support the fusion of recipient tissue cells is crucial for achieving a good clinical therapeutic effect after corneal implantation. Preventing the loss of active components of the extracellular matrix at the limbus of the transplanted cornea and constructing resistance-reducing channels for recipient tissue cell migration are key to achieving biointegration of the transplanted cornea with the recipient and improving post-transplant biostability. Based on this, the present invention provides the following technical solution: In a first aspect, the present invention provides a method for preparing a spoke-shaped decellularized cornea, the method comprising covering the upper and lower surfaces of a biological cornea with an insulating material, cutting the biological cornea covered with the insulating material using a corneal trephine, placing the cut material together with the corneal trephine in a low-temperature environment, and controlling the low-temperature field to diffuse from the periphery of the cornea to the center to construct spoke-shaped microchannels.

[0011] Specifically, the method includes the following steps: (a1) Cover the upper and lower surfaces of the biological cornea with heat-insulating material, cut the biological cornea covered with heat-insulating material using a corneal trephine, and place the cut material together with the corneal trephine in a low-temperature environment for 20-40 minutes. (a2) Remove and allow the cornea to fully thaw at room temperature; (a3) Repeat the freeze-thaw process of steps (a1) and (a2) three times; (a4) Transfer the cornea into a freeze-drying apparatus and freeze-dry for 12-24 hours to obtain a cornea with spoke-shaped microchannels.

[0012] In some embodiments of the present invention, the low-temperature environment is selected from a freezer with a temperature of -20°C to -80°C.

[0013] Preferably, the low-temperature environment is selected from a freezer with a temperature range of -40°C to -80°C.

[0014] The thermal insulation material described in this invention is a polymer material with low thermal conductivity, and the thermal conductivity of the thermal insulation material is preferably 0.02-0.04 W / (m·K).

[0015] In specific embodiments of the present invention, the polymer materials that can be selected for the thermal insulation material include, but are not limited to, polymer foam materials such as polyethylene, polypropylene, polyurethane, polyamide, polyimide, and polystyrene with micron or / and nanopore sizes.

[0016] In one specific embodiment of the present invention, the polymer material is polypropylene (PP) foam, which is circular in shape.

[0017] The thickness of the insulation material can be preset according to the specific temperature range of the low-temperature environment. Preferably, the thickness of the insulation material is ≥3 cm.

[0018] In a specific embodiment of the present invention, the heat insulation material is circular, and its diameter is 3-5 times the diameter of the cornea.

[0019] In this invention, the corneal trephine used for cutting the cornea is a commonly used instrument in ophthalmic surgery. The trephine is made of stainless steel and has high thermal conductivity. The shape and size of the trephine blade can be customized according to specific needs.

[0020] The technical principle of constructing spoke-shaped microchannels in the cornea in this invention is as follows: This invention reduces heat exchange between the upper and lower surfaces of the biological cornea by covering them with insulating material, leaving the periphery of the cornea exposed. A corneal trephine is used to cut the cornea covered with insulating material. While the trephine is in place, the exposed periphery of the cornea is in close contact with it. The cut material, along with the trephine, is placed in a low-temperature environment of -20°C to -80°C. Because the trephine is made of stainless steel, its thermal conductivity is higher, causing the periphery of the cornea in contact with the trephine to be the first to come into contact with the low-temperature environment, resulting in preferential ice crystal formation at the edge of the wet biological cornea. Since the insulating material significantly reduces heat exchange between the upper and lower surfaces of the cornea, the temperature at the center of the cornea is higher than that at the periphery. As the low-temperature field diffuses from the periphery to the center over time, the direction of ice crystal growth always extends towards the warmer central region of the cornea. The process of ice crystals diffusing from the periphery to the center of the cornea results in spoke-shaped crystals with an oriented structure, forming spoke-shaped microchannels within the cornea. The core purpose of implementing three freeze-thaw cycles is to strengthen the structural memory of the collagen fiber network for these spoke-shaped channels through repeated nucleation and maturation processes of ice crystals. The first freeze forms an initial columnar ice crystal template, whose growth path leaves stress tracks in the fiber network; during refreezing, the ice crystals dissolve while the fiber network retains its deformation due to stress relaxation; during the second freeze, water molecules preferentially re-nucleate along the original stress tracks, resulting in larger ice crystals with a more regular arrangement.

[0021] The biological cornea described in this invention is selected from xenogeneic cornea or allogeneic cornea. Xenogeneic cornea is animal-derived cornea, including but not limited to corneas from monkeys, pigs, cattle, sheep, horses, dogs, and rats. Allogeneic cornea includes but is not limited to corneas from humans or cadavers.

[0022] Furthermore, the method also includes the following steps: (b1) Rehydrate the cornea to construct spoke-shaped microchannels; (b2) The cornea obtained in step (b1) is decellularized to obtain a spoke-shaped decellularized cornea.

[0023] The decellularization methods include chemical methods (such as treatment with various surfactants such as Triton X-100, sodium dodecyl sulfate, and sodium deoxycholate), physical methods (high and low osmotic pressure cycling, repeated freeze-thaw cycles, and ultrastatic water pressure treatment to destroy cell membranes), and biological methods (such as treatment with various biological enzymes such as DNase, RNase, and trypsin) to decellularize biological corneas.

[0024] In some embodiments of the present invention, the decellularization method is as follows: the cornea to be treated is placed in a Triton X-100 solution with a mass fraction of 1-2% at an isocolloid osmotic pressure for 12-24 hours, during which time it is treated with ultrasound at 40 kHz and 480W power for 15 minutes every 4 hours, and then washed with distilled water 3-5 times to achieve decellularization.

[0025] In a preferred embodiment of the present invention, the specific decellularization method of step (b2) is as follows: the cornea is treated with an aqueous solution containing 4 wt% dextran (molecular weight of 80 kDa) and 2 wt% Triton X-100 for 24 hours by shaking, during which time it is treated with ultrasound at 40 kHz and 480 W power for 15 min every 4 hours, washed 3 times with isotonic dextran aqueous solution for 30 minutes each time, and placed in an isotonic dextran aqueous solution containing 1% double antibiotics for storage at 4°C for later use.

[0026] In a second aspect, the present invention provides a spoke-shaped decellularized cornea, characterized in that the cornea is prepared by the method described in the first aspect of the present invention.

[0027] Thirdly, the present invention provides the application of the spoke-shaped decellularized cornea in the preparation of corneal transplant products.

[0028] The advantages of the technical solution provided by this invention are as follows: This invention innovatively establishes a gradient temperature field from the periphery to the center of sheet-like biological corneal tissue. This allows the phase transition process of water molecules to proceed in an orderly manner along the temperature gradient direction, ensuring that the leading edge of ice crystal growth in the biological cornea always faces the warmer central region, preventing the irregular growth of ice crystals from the top and bottom surfaces. The orderly growth of ice crystals from the periphery to the center of the cornea forms spoke-shaped crystals with a directional structure. After the ice crystals melt, the spoke-shaped microchannels remain aligned with the temperature gradient direction, exhibiting structural order. The spoke-shaped directional microchannel structure in the cornea forms a low-resistance channel from the center to the periphery, providing a convenient pathway for the removal of cellular components from the central cornea. This significantly reduces the resistance to the removal of cellular components in the central region, accelerates the decellularization process, and prevents excessive decellularization in the peripheral cornea, which leads to excessive loss of extracellular matrix bioactive components. This invention systematically solves the problem of uneven corneal decellularization through the construction of directional channels. Furthermore, the channel forms a biomimetic migration path between the cornea and the recipient, which facilitates the migration and infiltration of recipient cells into the artificial cornea, reduces the migration resistance of tissue cells inside the cornea, promotes the biological healing of the transplanted cornea and the recipient corneal limbus, and improves the biological stability of the transplanted cornea. Attached Figure Description

[0029] Figure 1 A schematic diagram of the preparation method for spoke-shaped decellularized cornea.

[0030] Figure 2 Schematic diagram of the microstructure of corneal microchannels.

[0031] Figure 3 Scanning electron microscope image of a spoke-shaped decellularized cornea.

[0032] Figure 4 Image showing the decellularization effect of spoke-shaped decellularized cornea. Detailed Implementation

[0033] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. 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.

[0034] Example 1: Preparation of spoke-shaped decellularized cornea A schematic diagram of the preparation process of the spoke-shaped decellularized cornea is shown below. Figure 1 As shown, the specific operation steps are as follows: S1: Acquisition of xenograft corneas Taking pig cornea as an example, healthy adult pigs are obtained from professional breed pig farms. After the pigs are euthanized, fresh pig eyeballs are collected immediately and subjected to low-temperature preservation. In the laboratory, the epithelial cells are carefully scraped off with a scalpel, and the round or oval pig cornea is removed. The corneal surface is then briefly cleaned with water or PBS solution to remove any remaining dirt.

[0035] S2: Constructing spoke-shaped microchannels 2.1) Wipe the pig cornea thoroughly dry on absorbent paper to remove moisture from the corneal surface, and then place it between two cylindrical heat insulation materials (polypropylene (PP) foam), the diameter of which is 1.5 times the diameter of the cornea and the thickness is 3 cm. 2.2) After the cornea is stably placed between the insulation materials, the cornea covered with the insulation material is cut using a corneal trephine with a diameter of 10 mm. The cut cornea, along with the trephine, is placed in a low temperature environment of -80°C for 30 min to fully crystallize. The above system is then removed from the low temperature environment and placed at room temperature for 20 min to allow the crystals in the cornea to fully melt. The above freeze-thaw process is repeated three times. 2.3) The material from the insulation material in the aforementioned low-temperature environment is quickly removed and freeze-dried in a freeze dryer to enhance the stability of the constructed microchannels. A schematic diagram of the resulting spoke-shaped microchannels is shown below. Figure 2 As shown.

[0036] S3: Decellularization treatment 3.1) Rehydrate the cornea from which the spoke-shaped microchannels were constructed; 3.2) The cornea was treated with an aqueous solution containing 4 wt% dextran (molecular weight 80 kDa) and 2 wt% Triton X-100 for 24 hours with shaking. During this period, the cornea was treated with ultrasound at 40 kHz and 480 W power for 15 min every 4 hours. The cornea was washed three times with an isotonic dextran aqueous solution for 30 minutes each time. After that, the cornea was placed in an isotonic dextran aqueous solution containing 1% double antibiotics and stored at 4°C for later use.

[0037] Comparative Example 1: Preparation of radially decellularized cornea Step S1) The method for obtaining the allogeneic cornea is the same as in Example 1, except that in step S2), this comparative example uses heat-insulating materials and does not involve corneal trephine drilling. The specific operation method is as follows: S2: Constructing spoke-shaped microchannels 2.1) Wipe the pig cornea thoroughly dry on absorbent paper to remove moisture from the corneal surface, and then place it between two cylindrical heat insulation materials (polypropylene (PP) foam), the diameter of which is 1.5 times the diameter of the cornea and the thickness is 3 cm. 2.2) Place the above-mentioned insulation system in a low-temperature environment of -80℃ for 30 min to fully crystallize. Remove the system from the low-temperature environment and place it at room temperature for 20 min to allow the crystals in the cornea to fully melt. Repeat the above freeze-thaw process three times. 2.3) Quickly remove the material from the insulation material in the above low-temperature environment and freeze-dry it in a freeze dryer.

[0038] Step S3) The decellularization process is the same as in Example 1, and a spoke-shaped decellularized cornea is prepared and stored at 4°C for later use.

[0039] Comparative Example 2: Preparation of radially decellularized cornea Step S1) The method for obtaining the xenograft cornea is the same as in Example 1, except that in step S2), this comparative example uses a corneal trephine drill without any heat insulation material. The specific operation method is as follows: S2: Constructing spoke-shaped microchannels 2.1) Wipe the pig cornea thoroughly dry on absorbent paper to remove any moisture from the corneal surface; 2.2) Use a corneal trephine with a diameter of 10 mm to cut the cornea. Place the cut cornea along with the trephine in a low temperature environment of -80℃ for 30 min to fully crystallize. Remove the above system from the low temperature environment and place it at room temperature for 20 min to allow the crystals in the cornea to fully melt. Repeat the above freeze-thaw process three times. 2.3) Quickly remove the material from the insulation material in the above low-temperature environment and freeze-dry it in a freeze dryer.

[0040] Step S3) The decellularization process is the same as in Example 1, and a spoke-shaped decellularized cornea is prepared and stored at 4°C for later use.

[0041] Comparative Example 3: Preparation of radially decellularized cornea Step S1) The method for obtaining the xenograft cornea is the same as in Example 1, except that in step S2), this comparative example does not use insulating materials or corneal trephine. The specific operation method is as follows: S2: Constructing spoke-shaped microchannels 2.1) Wipe the pig cornea thoroughly dry on absorbent paper to remove any moisture from the corneal surface; 2.2) Place the cornea in a low temperature environment of -80℃ for 30 min to allow it to fully crystallize. Remove the cornea from the low temperature environment and place it at room temperature for 20 min to allow the crystals in the cornea to fully melt. Repeat the above freeze-thaw process three times. 2.3) Quickly remove the material from the insulation material in the above low-temperature environment and freeze-dry it in a freeze dryer.

[0042] Step S3) The decellularization process is the same as in Example 1, and a spoke-shaped decellularized cornea is prepared and stored at 4°C for later use.

[0043] Example 1: The effect of spoke-shaped channel construction on corneal microstructure Methods: The spoke-shaped decellularized corneas prepared in Example 1 and Comparative Examples 1-3 were quenched with liquid nitrogen and their cross-sectional structures were observed under a scanning electron microscope.

[0044] Result: As Figure 3 As shown, the cross-sectional image of Example 1 clearly shows a directional microchannel structure extending into the depth of the cornea. These channels continue to extend into the material, with smooth and continuous channel walls. This structural feature is due to the synergistic effect of the high thermal conductivity path provided by the stainless steel trephine and the radial temperature gradient formed by the thermal insulation material. Ice crystals grow directionally from the outside to the inside along the temperature gradient and are strengthened and fixed during three cycles of crystallization. Finally, after freeze-drying, the spoke-shaped channel system is formed.

[0045] Although some signs of inward-extending channels can be observed in the longitudinal section image of Comparative Example 1, the number of channels is significantly reduced, the continuity is poor, and the arrangement is disordered. The channel structure is blurred or even interrupted in some areas. This is because the lack of directional heat conduction effect of high thermal conductivity ring drills means that the insulation material alone cannot establish an effective radial temperature gradient. The nucleation and growth direction of ice crystals is highly random, resulting in disordered channel orientation and weak inward extension ability, which can only form local weakly directional pores.

[0046] The longitudinal section of Comparative Example 2 shows that the edge region is severely densified, and almost no effective channel structure extending inward can be observed. The hole wall is thick and the pores are closed. This is because the trephine directly contacts the edge, causing the region to cool down rapidly. At the same time, the central region also cools down rapidly due to the lack of heat insulation material. The overall temperature gradient is weak and unevenly distributed, making it impossible to form a directional ice crystal growth front. After freeze-drying, a completely non-directional sponge-like structure is obtained.

[0047] The longitudinal section of Comparative Example 3 also exhibits a typical disordered sponge-like structure, without any channel features extending inward in a directional manner. The pores are tortuous, closed, and isotropic, representing a typical result of traditional temperature-controlled phase separation methods.

[0048] In summary, only Example 1, through the combination of a high thermal conductivity ring drill and a low thermal conductivity insulation material, can construct an effective radial temperature gradient, driving ice crystals to grow directionally from the periphery to the center and forming spoke-shaped microchannels extending into the interior depth. Comparative Examples 1-3, lacking key structural features, could not achieve this directional structure, fully demonstrating the synergistic effect and significant technological progress of the technical solution of this invention.

[0049] Example 2: The effect of spoke-shaped channel construction on decellularization effect Figure 4 The images show the histological staining results (H&E staining) of the central region of the cornea after decellularization treatment. From left to right, they are fresh porcine cornea, Example 1, Comparative Example 1, Comparative Example 2, and Comparative Example 3. Through comparative analysis of the images, it can be clearly seen that the corneal material with spoke-shaped microchannels constructed in Example 1 exhibits excellent decellularization effect after decellularization treatment. There are very few residual cells in its stromal region, the collagen fibers are arranged regularly and the structure is intact, and the microchannel structure is clearly visible and remains unobstructed. This good decellularization effect is attributed to the effective mass transfer pathway provided by the microchannels, which allows the decellularization reagent to fully penetrate into the interior of the material and fully contact and react with the cellular components. The cellular components can directly exude through the microchannels.

[0050] In Comparative Example 1, the decellularization effect was mediocre, with a few scattered cell nuclei remaining in the matrix, indicating incomplete decellularization. This is directly related to the limited number, poor continuity, and disordered arrangement of microchannels in this group, leading to a tortuous reagent penetration path and limited mass transfer efficiency. In Comparative Example 2, due to the dense arrangement of the overall collagen structure of the material, the decellularization reagent and intracellular substances had difficulty effectively penetrating into and out of the porcine cornea. Images showed a large number of cellular components remaining in the matrix, indicating poor decellularization. Comparative Example 3 showed the worst decellularization effect, with many cell nuclei remaining in the matrix, failing to achieve effective decellularization. The layered collagen structure severely hindered the deep penetration of the reagent, and the cellular components in the internal region were basically preserved.

[0051] The above analysis fully demonstrates that the spoke-shaped microchannel structure constructed in Example 1 of this application significantly improves decellularization efficiency by providing an efficient material transport network. This structural advantage has key technical value in the preparation of tissue engineering scaffold materials. In contrast, Comparative Examples 1-3 failed to form effective directional mass transfer channels, resulting in poor decellularization effects. This further verifies the significant progress of the technical solution of this application in balancing structural orderliness and functional efficiency.

[0052] Those skilled in the art know that the central region of the cornea has a dense structure and low porosity. Achieving good decellularization in the central region requires high-concentration detergents or prolonged, repeated washing of the cornea. While potent surfactants offer better decellularization, the use of ionic, potent detergents like sodium deoxycholate often results in the loss of extracellular matrix active components and structural damage to the prepared peripheral corneal tissue. Although these methods effectively remove cells during the treatment of biological corneas, they inevitably disrupt the arrangement of extracellular matrix collagen fibers, leading to decreased mechanical properties and the loss of bio-inducible active substances.

[0053] The method for constructing microchannels in the cornea provided by this invention can more effectively support the exudation of cellular components inside the cornea. Under the same decellularization method and time conditions, the cornea prepared by the method for constructing microchannels provided in Example 1 has a better decellularization effect.

[0054] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a spoke-shaped decellularized cornea, characterized in that, The method includes covering the upper and lower surfaces of a biological cornea with an insulating material, cutting the biological cornea covered with the insulating material using a corneal trephine, placing the cut material together with the corneal trephine in a low-temperature environment, and controlling the low-temperature field to diffuse from the periphery of the cornea to the center to construct a spoke-shaped microchannel.

2. The preparation method according to claim 1, characterized in that, The method includes the following steps: (a1) Cover the upper and lower surfaces of the biological cornea with heat-insulating material, cut the biological cornea covered with heat-insulating material using a corneal trephine, and place the cut material together with the corneal trephine in a low-temperature environment for 20-40 minutes. (a2) Remove and allow the cornea to fully thaw at room temperature; (a3) Repeat the freeze-thaw process of steps (a1) and (a2) three times; (a4) Transfer the cornea into a freeze-drying apparatus and freeze-dry for 12-24 hours to obtain a cornea with spoke-shaped microchannels.

3. The preparation method according to any one of claims 1-2, characterized in that, The low-temperature environment is selected from a freezer with a temperature of -20℃ to -80℃; the insulation material is a polymer material with low thermal conductivity, which is 0.02-0.04 W / (m·K).

4. The preparation method according to claim 3, characterized in that, The insulation material is selected from high molecular foam materials such as polyethylene, polypropylene, polyurethane, polyamide, polyimide, and polystyrene with micron and / or nanopores. It is circular in shape, with a diameter of 3-5 times the diameter of the cornea and a thickness of ≥3 cm.

5. The preparation method according to any one of claims 1-2, characterized in that, The biological cornea is selected from xenogeneic cornea or allogeneic cornea.

6. The preparation method according to any one of claims 1-2, characterized in that, The method further includes the following steps: (b1) Rehydrate the cornea to construct spoke-shaped microchannels; (b2) The cornea obtained in step (b1) is decellularized to obtain a spoke-shaped decellularized cornea.

7. The preparation method according to claim 6, characterized in that, The decellularization method is selected from chemical, physical, or biological methods for decellularizing biological corneas; among them, chemical methods include treatment with various surfactants such as Triton X-100, sodium dodecyl sulfate, and sodium deoxycholate; physical methods include high and low osmotic pressure cycling, repeated freeze-thaw cycles, and ultrastatic water pressure treatment to destroy cell membranes; biological methods include treatment with DNase, RNase, and trypsin.

8. The preparation method according to claim 7, characterized in that, The specific decellularization method for step (b2) is as follows: The cornea is treated with an aqueous solution containing 4 wt% dextran (molecular weight of 80 kDa) and 2 wt% Triton X-100 for 24 hours by shaking. During this period, the cornea is treated with ultrasound at 40 kHz and 480 W power for 15 min every 4 hours. The cornea is washed 3 times with an isotonic dextran aqueous solution for 30 minutes each time. The cornea is then placed in an isotonic dextran aqueous solution containing 1% double antibiotics and stored at 4°C for later use.

9. A spoke-shaped decellularized cornea, characterized in that, The cornea is prepared by the method described in any one of claims 1-8.

10. The use of the spoke-shaped decellularized cornea according to claim 9 in the preparation of corneal transplant products.

Images