A decellularized limbal stroma and methods of making and using the same

Abstract

The application belongs to the technical field of bioengineering, and discloses a kind of acellular corneal limbus matrix and its preparation method and application, the preparation method of the acellular corneal limbus matrix includes the following steps: S1, natural corneal limbus tissue is subjected to acellular treatment, and acellular corneal limbus tissue is obtained;S2, amino acid is used to graft modification to the acellular corneal limbus tissue, and modified acellular corneal limbus tissue is obtained;S3, modified acellular corneal limbus tissue is placed into a solution containing heparan sulfate proteoglycan to carry out crosslinking reaction, and acellular corneal limbus matrix is obtained.The acellular corneal limbus matrix has similar hydration state and viscoelastic mechanical properties as natural corneal limbus tissue, realizes functional reconstruction of corneal limbus stem cell niche, and thus significantly improves the application potential of the scaffold material in stem cell maintenance and tissue regeneration.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a decellularized limbal matrix, its preparation method, and its application. Background Technology

[0002] The normal physiological function of the limbal tissue depends on the precise synergy of its unique hydration state, biomechanical properties, and niche microenvironment. Limbal stem cells (LSCs) are located in the three-dimensional niche structure of the corneal-scleral junction. Their growth depends on a favorable ecological environment of the limbus. LSCs generate transiently proliferating cells through asymmetric division, driving corneal epithelial regeneration and maintaining the transparency barrier function. The limbus has unique physiological and anatomical structures, including the "Vogt" palisade structure formed by fibrovascular ridges, while limbal crypts are groove-like structures located between the "Vogt" palisades. Limbal epithelial crypts and focal stromal protrusions are other compartments in this region, mainly located at the base of the "Vogt" palisades. The limbal epithelial crypts are protrusions from the subepithelial surface of the limbus into the stroma, serving as the direct environment for limbal stem cell contact. However, limbal stem cell deficiency (LSCD), caused by chemical / thermal burns, infections, immune diseases, etc., can lead to corneal epithelial defects, conjunctivalization, neovascularization, and persistent inflammation, resulting in vision impairment or blindness. This severely affects patients' work and life, and greatly increases the burden on patients' families and society.

[0003] According to the latest global consensus on the management of LSCD, patients with LSCD progressing to stage II or III require stem cell transplantation to reconstruct damaged corneal epithelium and restore the physiological barrier of the limbus and stem cell regeneration function. Currently, autologous / allogeneic limbal transplantation is the most direct and effective method for supplementing stem cell numbers and reconstructing damaged limbus. However, iatrogenic LSCD, scarcity of transplant materials, and immunosuppressants severely limit the implementation of this treatment. With the development of regenerative medicine, cultured limbal epithelial transplantation (CLET) and simple limbal epithelial transplantation (SLET) have become common stem cell treatment options for LSCD. However, numerous clinical studies report that the success rate of current CLET surgery is less than 80%, and the long-term success rate can drop to 50-60%; SLET is only suitable for unilateral eye treatment, with a short-term success rate of only 76%-84%. The reason for this is that simple LSC transplantation fails to repair the damaged natural physiological niche structure and components of the limbus. Therefore, the development of transplantation materials with natural limbal physiological ultrastructure and components has significant clinical and social value.

[0004] The technical approaches for fabricating scaffold materials with limbal physiological ultrastructures are mainly divided into artificial niche synthetic scaffolds and naturally derived scaffolds. Artificial niche synthetic scaffolds are fabricated primarily using substrates such as collagen hydrogels and polymer materials, employing high-precision molds, micro-stereolithography, electrospinning, and 3D printing to create synthetic scaffold materials with limbal crypt-like ultrastructures. However, due to limitations in processing precision, existing synthetic scaffolds still lag significantly behind the physiological levels of the natural limbus and meet transplantation requirements in terms of extracellular matrix, ultrastructure, and biomechanical properties.

[0005] In naturally derived scaffolds, decellularized matrix materials are the primary technological approach. By selecting xenogeneic limbal tissue and removing xenogeneic cells through decellularization, a decellularized limbal matrix containing natural tissue structures such as limbal crypts, focal stromal protrusions, and vascular networks can be obtained. However, it is worth noting that natural collagen fibers are connected by a large number of soluble proteoglycans, which participate in maintaining the orientation of collagen fibers through intermolecular glycosidic bonds, hydrogen bonds, and electrostatic forces. These proteoglycans are also important attachment sites for growth factors in the natural matrix. Studies have found that various surfactants, digestive enzymes, or physical actions during decellularization can damage these soluble components and ultrastructures, reducing the number of hydrophilic groups in the decellularized matrix, decreasing biomechanical strength, and causing a significant loss of growth factors. Only 50%-80% of natural collagen and 10%-30% of glycosaminoglycans can be retained, which affects the activity, tissue regeneration, and physiological function of implanted limbal stem cells, thus greatly reducing the therapeutic value of decellularized limbal scaffolds. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing decellularized limbal matrix, wherein the prepared decellularized limbal matrix retains the unique three-dimensional niche structure of the limbus and has a hydration state and viscoelastic mechanical properties similar to those of natural limbal tissue.

[0007] The following technical solutions are used to achieve the above objectives.

[0008] In a first aspect, the present invention provides a method for preparing decellularized limbal stroma, comprising the following steps:

[0009] S1. Natural limbal tissue is decellularized to obtain decellularized limbal tissue;

[0010] S2. The decellularized limbal tissue is grafted with amino acids to obtain modified decellularized limbal tissue;

[0011] S3. The modified decellularized limbal tissue is placed in a solution containing heparan sulfate proteoglycan to undergo a cross-linking reaction, thereby obtaining a decellularized limbal matrix.

[0012] In some embodiments, in step S3, the heparan sulfate proteoglycan is selected from one or more of syndecan, glypican, perlecan, and agarin, preferably agarin.

[0013] In some embodiments, the ratio of the modified decellularized limbal tissue to the heparan sulfate proteoglycan is 0.1g:1μg~15μg, preferably 0.1g:1μg~10μg, more preferably 0.1g:2μg~8μg, and even more preferably 0.1g:4μg~8μg.

[0014] In some embodiments, in step S3, the reaction temperature is 2°C to 8°C and the time is 20h to 28h.

[0015] In some embodiments, in step S2, the amino acid is selected from at least one of aspartic acid, glutamic acid, 2-aminoadipic acid, cystine, homocysteine, S-carboxymethyl-L-cysteine, and 4-methylglutamic acid, preferably aspartic acid.

[0016] In some embodiments, in step S2, the mass ratio of the decellularized limbal tissue to amino acids is 0.1:5~15.

[0017] In some embodiments, in step S2, the amino acids react with the decellularized limbal tissue in a crosslinking solution; preferably, the reaction is performed under ultrasound for 1 h to 8 h at a power of 10 W to 100 W; and / or, the crosslinking solution comprises a 0.02 to 0.012 mmol / L N-hydroxysuccinimide solution and a 0.02 to 0.012 mmol / L 1-ethyl-(3-dimethylaminopropyl)carbodiimide solution, wherein the decellularized limbal tissue is first treated with the N-hydroxysuccinimide solution for 5 h to 30 h, and then treated with a mixed solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and amino acids for 1 h to 8 h.

[0018] Furthermore, it should be noted that the grafting modification reaction of the amino acid onto the decellularized limbal tissue can also be carried out under other conditions, such as at least one of chemical cross-linking agents, enzymatic cross-linking agents, and physical reactions.

[0019] The chemical crosslinking agent includes at least one of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, N-hydroxysuccinimide, 1,1-carbonyldiimidazole, genipin, proanthocyanidins, ethylene glycol diglycidyl ether, and glutaraldehyde.

[0020] The enzymatic cross-linking agent includes at least one of horseradish peroxidase, hydrogen peroxide, and glutamine transferase.

[0021] The physical reaction includes at least one of physical blending, ultraviolet crosslinking, thermal dehydrogenation crosslinking, and ionic crosslinking.

[0022] In some embodiments, in step S1, the natural limbal tissue is placed in a decellularization solution for decellularization treatment; the decellularization solution comprises the following components: phospholipase 15 ml / L to 25 ml / L, sodium deoxycholate 8 g / L to 12 g / L, NaCl 5 g / L to 8 g / L, KCl 0.3 g / L to 0.6 g / L, CaCl2 0.1 g / L to 0.4 g / L, MgCl2·6H2O 0.1 g / L to 0.3 g / L, glucose 0.5 g / L to 1.5 g / L, NaHCO3 2 g / L to 5 g / L, antibacterial agent 8 ml / L to 15 ml / L, and the remainder is water.

[0023] In addition, it should be noted that the natural limbal tissue can also be treated using other decellularization methods, such as enzymatic digestion to destroy the cell structure within the natural limbal tissue; chemical methods to destroy the cell structure within the natural limbal tissue using chemical reagents; and physical methods to destroy the cell structure within the natural limbal tissue.

[0024] The enzyme includes at least one of phospholipase, protease, deoxyribonuclease, and nuclease;

[0025] The chemical reagent includes at least one of surfactants, acids, bases, and chelating agents;

[0026] The physical method includes at least one of the following: oscillation, ultrasound, osmotic pressure change, supercritical fluid, and repeated dynamic melting.

[0027] In some embodiments, the decellularization treatment is carried out at a temperature of 35-38°C for 4-12 hours.

[0028] And / or, the decellularization process is carried out in a shaker at a speed of 100-1000 r / min;

[0029] And / or, the antibacterial agent includes penicillin, streptomycin, and amphotericin B; preferably, the concentration of penicillin is 80 U / ml to 120 U / ml, the concentration of streptomycin is 80 μg / ml to 120 μg / ml, and the concentration of amphotericin B is 0.1 μg / ml to 0.3 μg / ml.

[0030] In some embodiments, the natural limbal tissue is xenogeneic, allogeneic, or autologous natural limbal tissue, preferably porcine natural limbal tissue.

[0031] In a second aspect, the present invention provides a decellularized limbal matrix prepared by the method described above.

[0032] In a third aspect, the present invention provides the use of the decellularized limbal matrix as described above in the preparation of limbal epithelial stem cell products.

[0033] In this invention, decellularized limbal tissue after appropriate decellularization treatment is first modified by grafting with amino acids, and then cross-linked with specific proteoglycans. This yields a decellularized limbal matrix cross-linked with amino acids and proteoglycans. By optimizing the proteoglycan concentration, immunogenic components in the limbal tissue can be efficiently removed, and the limbal niche can be effectively restored in both physical structure and composition. It also possesses a hydration state and viscoelastic mechanical properties similar to natural limbal tissue, achieving functional reconstruction of the limbal stem cell niche. This enables implanted limbal stem cells to maintain long-term homeostasis and renewal, as well as maintain cell stemness, thereby significantly enhancing the application potential of scaffold materials in stem cell maintenance and tissue regeneration. Attached Figure Description

[0034] Figure 1 AG is a histological and component analysis of NPLS and APLS; among which Figure 1 A shows the HE staining results of APLS and NPLS; Figure 1 B shows the results of APLS and NPLS staining of cell nuclei; Figure 1 C represents the results of APLS and NPLS alicin blue staining; Figure 1 D represents the periodic acid-Schiff staining results from APLS and NPLS; Figure 1 E represents the results of APLS and NPLS DNA quantification analysis; Figure 1 F represents the quantitative analysis results of hydroxyproline by APLS and NPLS; Figure 1 G represents the quantitative analysis of glycosaminoglycans by APLS and NPLS; H represents the cytotoxicity test results of APLS extract (n=5) (*P<0.05, ****P<0.0001).

[0035] Figure 2 AD is an ultrastructural analysis method combining NPLS and APLS; among which Figure 2 A is a SEM image of the corneal-limbal junction from APLS and NPLS. * represents the corneal region, # represents the limbal region, and the dashed line is the junction line between the cornea and the limbus. Figure 2 B is the SEM image from APLS ( Figure 2 (Enlarged image within the black box A); Figure 2 C represents the TEM results for APLS and NPLS niche structures; Figure 2 D represents the TEM results for APLS and NPLS collagen fibers.

[0036] Figure 3 AJ represents the chemical structure and water content detection results of Agrin crosslinked on APLS using ASP as the crosslinking arm; among which... Figure 3 A represents the FTIR results analysis of NPLS, APLS, APLS-ASP, and APLS-ASP-Agrin. The arrows indicate the bands of change between different groups: 3315 cm⁻¹ -1 The tensile vibration bands representing hydroxyl (-OH) and amino (-NH) groups; 2960 cm⁻¹ -1 and 2910cm -1 This represents the stretching vibration band of the -OH group in the carboxyl group; 1660 cm⁻¹ -1 Represents the carbonyl group (-C=O) in the amide bond; 1240cm -1 and 1080cm -1 It is a carbon-nitrogen bond (-CN); Figure 3 B is the immunofluorescence staining of agrin in NPLS, APLS, and APLS-ASP-Agrin; Figure 3 C represents the XRD results analysis of NPLS, APLS, APLS-ASP, and APLS-ASP-Agrin; Figure 3 D represents the XPS results analysis of NPLS, APLS, APLS-ASP, and APLS-ASP-Agrin; Figure 3 E is the size of the contact angle in each bracket; Figure 3 F) represents the change in water content of NPLS, APLS, APLS-ASP, and APLS-ASP-Agrin over time under ultrasonic power conditions of pH=2.79 and 100W (n=5). Figure 3 G is the side thickness diagram of NPLS, APLS, APLS-ASP, and APLS-ASP-Agrin after 24 hours of rehydration (n=5); Figure 3 H is the proportion of bound water in the scaffold (n=5); Figure 3 I is the proportion of free water in the stent (n=5); Figure 3 J represents the DSC curve results for each stent (n=5);

[0037] (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

[0038] Figure 4 AD represents the rheological properties of the scaffold; where Figure 4 A is the energy storage modulus of NPLS, APLS, APLS-ASP, and APLS-ASP-Agrin; Figure 4 B is the loss modulus of NPLS, APLS, APLS-ASP, and APLS-ASP-Agrin; Figure 4 C is the complex modulus of NPLS, APLS, APLS-ASP, and APLS-ASP-Agrin; Figure 4 D is the loss tangent of NPLS, APLS, APLS-ASP, and APLS-ASP-Agrin, P<0.05 (n=5).

[0039] Figure 5 This describes the effect of different Agrin concentrations on cell proliferation activity.

[0040] Figure 6 AC is an Agrin graft construction; where Figure 6 A is a diagram of APLS and the APLS-ASP-Agrin stent (HE diagram); Figure 6 B is a Hoechst staining image of APLS and the APLS-ASP-Agrin scaffold; Figure 6 C represents the immunofluorescence expression of P63 and CK3 in APLS and APLS-ASP-Agrin scaffolds.

[0041] Figure 7 AC is a graph showing the rehydration water content results of APLS-ASP-Agrin and APLS-Agrin; where Figure 7 A represents the proportion of free water. Figure 7 B is the bound water ratio. Figure 7 C represents the moisture content at different time points. Detailed Implementation

[0042] To facilitate understanding of the present invention, a more complete description will be provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0043] Unless otherwise specified, experimental methods in the following examples are generally performed under standard conditions or as recommended by the manufacturer. All commonly used chemical reagents used in the examples are commercially available products.

[0044] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this invention includes any and all combinations of one or more of the associated listed items.

[0045] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the embodiments thereof. 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 of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0046] Agrin is a high molecular weight heparan sulfate proteoglycan (HSPG) with a core protein molecular weight of approximately 200–220 kDa, which can reach over 400 kDa after different splicing variants and glycosylation modifications.

[0047] APLS: Decellularized porcine limbal tissue.

[0048] APLS-ASP: Decellularized porcine limbal tissue cross-linked with Asp.

[0049] APLS-ASP-Agrin: Decellularized porcine limbal stroma that crosslinks Asp and Agrin.

[0050] NPLS: Natural pig limbus.

[0051] Sources of materials and reagents

[0052] 1. Agrin:

[0053] The Agrin protein was purchased from MCE (MedChemExpress, USA) and is a recombinant human Agrin protein with a core protein molecular weight of approximately 210 kDa.

[0054] 2. EDC / NHS:

[0055] 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, molecular weight 191.70 Da, purchased from Sigma-Aldrich).

[0056] N-hydroxysuccinimide (NHS, molecular weight 115.09 Da, purchased from Sigma-Aldrich).

[0057] 3. Aspartic acid (Asp):

[0058] Aspartic acid (Asp, molecular weight 133.10 Da, purchased from Sigma-Aldrich).

[0059] 4. Phospholipase:

[0060] Phospholipase (molecular weight approximately 14–18 kDa, purchased from Sigma-Aldrich).

[0061] 5. Sodium deoxycholate:

[0062] Sodium deoxycholate (molecular weight 414.55 Da, purchased from Sigma-Aldrich).

[0063] 6. DNase I:

[0064] DNase I (molecular weight approximately 31 kDa, purchased from Roche or Sigma-Aldrich).

[0065] 7. Albumin:

[0066] Bovine serum albumin (BSA, molecular weight approximately 66.5 kDa, purchased from Sigma-Aldrich).

[0067] 8. Proanthocyanidins:

[0068] Proanthocyanidins (average molecular weight 500–3000 Da (mixture), purchased from Sigma-Aldrich).

[0069] 9. Perlecan:

[0070] Perlecan protein (molecular weight approximately 400–470 kDa, purchased from R&D Systems).

[0071] Example 1

[0072] (1) Preparation of decellularized porcine limbal tissue:

[0073] Fresh pig eyeballs were obtained from the slaughterhouse and rinsed three times with ultrapure water. Surgical instruments were used to remove the muscle tissue and conjunctiva of the pig eyeballs, and the corneal and scleral tissue was circumferentially cut about 5 mm outside the limbus, with a tissue diameter of about 18 mm. After removing the attached vitreous body, retina, and iris tissue, the eyeballs were rinsed five times with ultrapure water.

[0074] At -80℃, the treated porcine scleral tissue was placed in a serum bottle containing PBS solution and frozen for 2 hours. Then, it was shaken at 220 rpm for 2 hours in a constant temperature water bath at 37℃. This freeze-thaw cycle was repeated a total of 3 times to remove epithelial tissue. The porcine scleral tissue was then placed in pure water supplemented with penicillin (100 U / ml + streptomycin 100 μg / ml) and incubated overnight at 4℃ to allow the cells to absorb water, swell, and rupture, yielding the decellularized limbal tissue.

[0075] The following decellularization solution was used to treat the limbal tissue to be decellularized: At 15°C, the limbal tissue to be decellularized was immersed in the decellularization solution, which consisted of 1000 ml of pure water + 6.136 g NaCl + 0.41 g KCl powder + 0.1997 g CaCl2 + 0.1624 g MgCl2·6H2O + 0.9008 g glucose powder + 2.9403 g NaHCO3 powder, 20 ml phospholipase, 10 g sodium deoxycholate, and 10 ml of triple antibiotics (penicillin 100 U / ml + streptomycin 100 μg / ml + amphotericin B 0.25 μg / ml), with a solution pH of 8. Five limbal tissue samples were soaked in 600 mL of decellularization solution and shaken in a 37°C water bath for 12 hours. At 4°C, the tissues were transferred to PBS solution containing 1% penicillin (100 U / ml + streptomycin 100 μg / ml), and the samples were shaken and washed 40 times. The solution was changed every 3 hours to obtain decellularized porcine limbal tissue (APLS).

[0076] We found that APLS treated with appropriate decellularization still retained a high collagen content, and the tissue matrix did not change significantly, and the limbal crypt structure was relatively intact.

[0077] The tissue structure of both NPLS (natural porcine limbal tissue) and APLS (decellularized porcine limbal tissue) was examined. Results are as follows: Figure 1 As shown, compared with NPLS, APLS completely removes limbal epithelial stem cells, while the superficial limbal stroma layer remains intact, and collagen fibers are still neatly arranged. Figure 1 A) Hydroxyproline retention is as high as 85.5% ( Figure 1 F). Hoechst staining failed to detect nuclear fluorescence signals (F). Figure 1 B), DNA extraction and quantitative detection revealed that the DNA removal rate in APLS was as high as 92.3% ( Figure 1 E). Alicin blue and PAS staining showed that, compared with NPLS, the alicin blue and PAS staining in APLS scaffolds were weaker ( Figure 1 C& Figure 1D), but APLS still retains 40.3% of the glycosaminoglycan content ( Figure 1 G). To investigate whether the extract of decellularized tissue would have a toxic effect on cells, limbal stem cells were cultured in the extract. It was found that the cell growth rate in the extract group was consistent with that of normally cultured cells, and after five days of culture, there was no significant difference in cell number between the two groups. Figure 1 H).

[0078] Figure 2 This involves ultrastructural analysis of NPLS and APLS. SEM results show that the corneal morphology of NPLS and APLS is relatively smooth, with obvious protrusions and depressions in the limbus region. Figure 2 A). Magnifying the APLS image revealed that it retained a relatively complete limbal crypt structure. Figure 2 B). TEM analysis showed that, compared with NPLS, the cellular structure was completely removed in APLS, and the matrix did not change significantly. Figure 2 C). Furthermore, APLS collagen fibers are regularly arranged, and their diameter and density are well preserved. Figure 2 D)

[0079] (2) Cross-linking modification of decellularized porcine limbal tissue:

[0080] Using surgical scissors, cut equal portions of porcine limbal tissue from the prepared acellular porcine limbal tissue, one portion representing the cornea and the other the sclera. This acellular porcine limbal tissue was then immersed in 120 ml of 0.08 mmol / L NHS (N-hydroxysuccinimide) solution and activated in an ultrasonic cleaner for 15 minutes at 100 W. Subsequently, approximately 0.1 g of the acellular porcine limbal tissue was immersed in a mixed solution of 120 ml of 0.04 mmol / L EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride) and 6 mg / ml aspartic acid (Asp) for 5 hours at 100 W. Afterward, the tissue was removed at 4°C and thoroughly washed in ultrapure water for 20 hours. It was then placed in an air shaker at 80 rpm to obtain cross-linked Asp acellular porcine limbal tissue (APLS-ASP).

[0081] At 4°C, decellularized porcine limbal tissue slices cross-linked with Asp were placed in a 4 μg / ml Agrin (combined proteoglycan) solution (the Agrin solution was prepared by diluting the stock solution with double-distilled water). Each tissue slice (approximately 0.1 g) with a diameter of about 18 mm was added to 1 ml of Agrin solution and soaked for 24 hours to obtain decellularized porcine limbal matrix (APLS-ASP-Agrin) cross-linked with Asp and Agrin.

[0082] The prepared acellular porcine limbal stroma (APLS-ASP-Agrin) was used to extract circular tissue samples, each comprising half of the cornea and half of the sclera, using a 6.5 mm diameter corneal trephine for testing. The test results are shown below:

[0083] FTIR results showed that, compared with APLS, APLS-ASP was at 3315 cm⁻¹ -1 The tensile vibration bands of hydroxyl (-OH) and amino (-NH) groups at 1660 cm⁻¹ -1 The stretching vibration band of the carbonyl group (-C=O) in the amide bond is enhanced; 1240 cm -1 and 1080 cm -1 The carbon-nitrogen bond (-CN) characteristic band of the amino group is also enhanced. These changes indicate that APLS-ASP has a richer number of carboxyl and amino groups compared to APLS, suggesting that ASP has been successfully crosslinked onto APLS. Figure 3 A). Furthermore, compared to APLS-ASP, APLS-ASP-Agrin material at 2960 cm⁻¹… -1 2910 cm -1 The carbonyl (-C=O) characteristic band of the amide bond is enhanced at 1240 cm⁻¹. -1 The weakening of the characteristic band peak of the carbon-nitrogen bond (-CN) at the amino group indicates that Agrin successfully crosslinked onto the APLS-ASP scaffold through the amino group reaction. Figure 3 A). Immunofluorescence results showed that the epithelial structure of NPLS contained a large amount of Agrin protein. After decellularization, APLS did not contain Agrin protein, while APLS-ASP-Agrin showed obvious red fluorescence, confirming that Agrin was successfully cross-linked onto APLS. Figure 3 B).

[0084] XRD results showed that NPLS, APLS, APLS-ASP, and APLS-ASP-Agrin all exhibited the typical broad diffusion diffraction characteristics of the adhesive raw material, with no new sharp crystalline phase peaks observed, indicating that decellularization and subsequent crosslinking did not change the main crystalline phase composition of the material. Compared to NPLS, the overall diffraction intensity of APLS decreased, suggesting a decrease in matrix order after decellularization. The intensity partially recovered after ASP crosslinking, and further enhancement of the intensity after the introduction of Agrin indicated that crosslinking can improve the stability and compactness of the matrix structure. Figure 3 C).

[0085] C1s, O1s, and N1s characteristic peaks were detected in the full XPS spectrum. The surface elemental signal distribution changed after decellularization; the O1s and N1s signals of APLS-ASP were enhanced after ASP crosslinking, indicating an increase in oxygen- and nitrogen-containing functional groups on the surface; even after immobilizing Agrin, significant N1s and O1s peaks were still maintained, indicating that Agrin can be stably introduced and immobilized on the material surface through an ASP re-crosslinking strategy. Figure 3 D).

[0086] To further evaluate the performance of the four scaffolds, we measured the contact angle of each scaffold. The contact angles of APLS-ASP and APLS-ASP-Agrin were significantly smaller than those of APLS, and the contact angle of APLS-ASP-Agrin was closer to that of NPLS. This indicates that the surfaces of APLS-ASP and APLS-ASP-Agrin are more hydrophilic than those of APLS. Figure 3 E).

[0087] The rehydration water content of each stent was then tested at 10 min, 30 min, 2 h, and 24 h. The results showed that the water content of the stents gradually increased over time, reaching full rehydration after 24 hours. Compared with the APLS group, the APLS-ASP and APLS-ASP-Agrin stents exhibited significantly increased rehydration rates and final water content, with a smaller difference in final water content compared to the NPLS group. Figure 3 F) has a hydration state similar to that of NPLS.

[0088] Measurements of the thickness of the four types of stents revealed that, compared to APLS, APLS-ASP and APLS-ASP-Agrin showed significantly improved thicknesses, approaching those of NPLS. Figure 3 G).

[0089] The ratio of free water to bound water in the four stents was then calculated using DSC curves. The results showed that the ratios of free water to bound water in APLS-ASP and APLS-ASP-Agrin were higher than those in APLS. Compared with APLS, APLS-ASP and APLS-ASP-Agrin showed smaller differences compared with NPLS. Figure 3 H- Figure 3 J).

[0090] Viscoelasticity analysis of four scaffolds revealed that, within the frequency range of 0.1-10 Hz, the storage modulus and complex modulus of NPLS increased slightly with increasing frequency, while the loss modulus decreased in the 0.1-1 Hz range and increased in the 1-10 Hz range. Compared to NPLS, the decellularization process significantly increased these three moduli in APLS. Crosslinking with ASP-Agrin could reverse this trend to some extent, resulting in limbal stroma viscosity and elasticity more closely resembling those of normal limbus. Figure 4 A- Figure 4 C).

[0091] Although the loss tangent of APLS-ASP-Agrin and APLS-ASP differs more from that of NPLS compared to APLS ( Figure 4 D), but mainly because APLS-ASP and APLS-ASP-Agrin have a viscosity closer to NPLS than APLS, while their elastic properties are not reduced proportionally.

[0092] The prepared decellularized porcine limbal matrix (APLS-ASP-Agrin) slides were dried in a 40℃ constant temperature drying oven for 12 hours until constant weight. The matrix slides were packaged in sealed bags, labeled, sealed in aluminum foil bags, sterilized by cobalt-60 (25 kGey) irradiation, and stored at -20℃.

[0093] Example 2

[0094] 1.1 Rabbits were euthanized by air embolism.

[0095] 10 ml of air was injected through the marginal ear vein, and the rabbit's breathing, lip color, and pupil changes were then observed to confirm death.

[0096] 1.2 Rabbit corneal tissue sampling

[0097] 1) Use forceps to grasp the palpebral conjunctiva of the New Zealand rabbit, and then use ophthalmic scissors to cut along the eye socket to cut open the membrane and muscle surrounding the eyeball. Remove the rabbit eyeball and put it into a 50ml centrifuge tube containing 30ml of 1% penicillin (100U / ml + streptomycin 100μg / ml) pre-cooled phosphate buffered solution (PBS). Store at 4°C and collect corneal tissue within 2 hours.

[0098] 2) Shake vigorously in the centrifuge tube three times for 1 minute each time, and replace with fresh pre-cooled PBS after each wash.

[0099] 3) Then place the eyeball in a 6cm diameter culture dish, remove the excess tissue around the outside of the eyeball with tissue scissors, cut off most of the sclera along the corneal limbus and remove the contents, and scrape off the iris and other parts. Place it in a 50ml centrifuge tube containing clean PBS.

[0100] 4) Finally, wash three times with pre-cooled PBS containing 30ml of 1% penicillin (100U / ml penicillin + 100μg / ml streptomycin), 10 minutes each time.

[0101] 1.3 Primary culture of LSC

[0102] 1) Wash the cornea three times with PBS containing 1% penicillin (100 U / ml + streptomycin 100 μg / ml), for 1 minute each time.

[0103] 2) Use a trephine to drill a 3 mm diameter limbal tissue. Dip the limbal tissue in a small amount of fetal bovine serum (gibco) and place it in a six-well plate with the epithelial layer facing up. Place one limbal tissue in each well.

[0104] 3) Place at 37°C for 1 hour to allow it to adhere to the wall, until the fetal bovine serum around the tissue block is basically dry.

[0105] 4) Then add 2ml of KCM ((DMEM F10 + 10% fetal bovine serum + 1% penicillin 100U / ml + streptomycin 100μg / ml) + EGF 10ng / ml + human insulin 5mg / ml + transferrin 5mg / ml + hydrocortisone 4μg / ml + glutamine 5ml + MEM 5ml) and culture in a CO2 incubator, changing the medium every other day.

[0106] 5) Primary rabbit limbal stem cells (LSCs) were obtained 10 days later and used for subsequent experiments.

[0107] 1.4 Primary rabbit limbal stem cell seeding experiment

[0108] 1) Primary rabbit limbal stem cells were seeded into 96-well plates and divided into blank group, control group and different concentrations of Agrin stimulation group, with 6 replicates in each group, and cultured at 37℃ for 24h.

[0109] 2) After all cells have adhered to the culture vessel, add different concentrations (0.25ug / ml, 0.5ug / ml, 1ug / ml, 2ug / ml, 4ug / ml, 8ug / ml) of Agrin (MCE) medium and continue culturing for 24 h; the treatment without Agrin solution is used as the control group.

[0110] 3) After the culture is complete, discard the supernatant and add 88 μl of CCK8 detection solution to each well. The CCK8 detection solution is prepared as follows: 8 μl of CCK8 reagent (APExBIO) + 80 μl of complete culture medium; incubate at 37°C in the dark for 1 h.

[0111] 4) Detection at 450nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0112] The results are as follows Figure 5 The results showed that, compared with the control group, Agrin concentrations of 1 μg / ml to 8 μg / ml significantly enhanced the proliferative activity of seeded limbal stem cells, while the proliferative effect was not significantly different below 1 μg / ml. Therefore, when Agrin is absent or insufficient in the niche microenvironment, it will lead to a decrease in the local signaling molecule enrichment capacity and blockage of mechanotransduction, resulting in a series of functional changes such as weakened limbal stem cell proliferation, increased apoptosis levels, and enhanced abnormal differentiation tendency. On the other hand, the presence of specific concentrations of Agrin and its stable expression help maintain the survival, self-renewal, and functional stability of limbal stem cells, thereby ensuring that the limbal stem cell population is in a controlled and reversible homeostatic equilibrium in the long term.

[0113] Example 3

[0114] The primary rabbit limbal stem cells extracted in Example 2 were seeded onto APLS and APLS-ASP-Agrin prepared in Example 1, respectively. After culturing for 5 days, the tissues were fixed and embedded, and then sectioned.

[0115] 1) After dewaxing and rehydration, the sections are placed in boiling antigen retrieval solution (Solepro C1032) and microwaved on medium heat for 15 minutes, heating for 3 minutes each time and stopping for 1 minute.

[0116] 2) After the antigen retrieval solution has cooled to room temperature, remove the slide.

[0117] 3) Add 0.01% Triton solution to the tissue for 30 min to break the membrane.

[0118] 4) Circle the tissue with an immunohistochemical pen (to avoid the liquid from flowing and spreading during staining), and add blocking goat serum for one hour.

[0119] 5) After blocking, aspirate the serum from the tissue, dilute the primary antibody p63 and CK3 at a ratio of 1:200, and add PBS to the negative control group. Place the mixture in a humidified chamber with added water and incubate overnight at 4°C.

[0120] 6) Recover the primary antibody and wash three times with PBS solution for 10 min each time.

[0121] 7) Add the corresponding species secondary antibody to each tissue slice, cover it, and incubate at room temperature in the dark for 2 hours.

[0122] 8) Recover the secondary antibody and wash three times with PBS solution for 10 min each time.

[0123] 9) Dilute Hoechst with PBS at a ratio of 1:500, add Hoechst staining solution to the slide and stain for 10 min, then wash with PBS solution 3 times, 10 min each time.

[0124] 10) Place one or two drops of anti-quenching mounting medium on the tissue surface, mount the slide, gently cover it with a coverslip, and observe and photograph it using a laser confocal fluorescence microscope.

[0125] The results showed that after cross-linking with Agrin, the connections between cells and the matrix, as well as between cells, were relatively tight. Figure 6 A- Figure 6 B). Immunofluorescence staining of limbal grafts with P63 (a marker of stemness) and CK3 (a marker of differentiation) showed that grafts without Agrin crosslinking had fewer stem cells and more cells exhibiting a differentiated phenotype. After Agrin crosslinking, not only did most cells in the stromal contact area maintain the stemness of limbal stem cells, but CK3 signaling was also mainly expressed in the surface layer of stratified cells. This largely restored the physiological structure of the natural cornea. Figure 6 C).

[0126] Example 4

[0127] (1) The preparation of decellularized porcine limbal tissue was the same as in Example 1.

[0128] (2) The cross-linking modification of decellularized porcine limbal tissue differs from that in Example 1 in that: the grafting modification of aspartic acid (Asp) is omitted; only Agrin is cross-linked into the decellularized porcine limbal matrix.

[0129] The other components and preparation steps are the same as in Example 1.

[0130] The rehydration water content of APLS-Agrin and APLS-ASP-Agrin stents was tested at 10 min, 30 min, 2 h, and 24 h. The results showed that the water content of the stents gradually increased over time, reaching full rehydration after 24 hours. Compared with the APLS-Agrin group, the rehydration rate and final water content of the APLS-ASP-Agrin stent were significantly increased. Figure 7 C).

[0131] We then calculated the ratio of free water to bound water in the four stents using DSC curves. The results showed that the ratio of free water to bound water in APLS-ASP-Agrin was higher than that in APLS-Agrin. Figure 7 A, Figure 7B).

[0132] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0133] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A method for preparing decellularized limbal stroma, characterized in that, Includes the following steps: S1. Natural limbal tissue is decellularized to obtain decellularized limbal tissue; S2. The decellularized limbal tissue is grafted with amino acids to obtain modified decellularized limbal tissue; S3. The modified decellularized limbal tissue is placed in a solution containing heparan sulfate proteoglycan to undergo a cross-linking reaction, thereby obtaining a decellularized limbal matrix.

2. The preparation method according to claim 1, characterized in that, In step S3, the heparan sulfate proteoglycan is selected from one or more of multiligand proteoglycans, glycosylphosphatidylinositol-anchored proteoglycans, beaded proteoglycans, and combinatorial proteoglycans, preferably combinatorial proteoglycans.

3. The preparation method according to claim 2, characterized in that, The ratio of the modified decellularized limbal tissue to the heparan sulfate proteoglycan is 0.1g:1μg~15μg, preferably 0.1g:1μg~10μg, more preferably 0.1g:2μg~8μg, and even more preferably 0.1g:4μg~8μg.

4. The preparation method according to any one of claims 1-3, characterized in that, In step S2, the amino acid is selected from at least one of aspartic acid, glutamic acid, 2-aminoadipic acid, cystine, homocysteine, S-carboxymethyl-L-cysteine, and 4-methylglutamic acid, preferably aspartic acid.

5. The preparation method according to any one of claims 1-3, characterized in that, In step S2, the mass ratio of the decellularized limbal tissue to amino acids is 0.1:5~15; And / or, the amino acids react with the decellularized limbal tissue under the action of a cross-linking agent; preferably, the cross-linking agent is composed of N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide; And / or, the reaction is treated with ultrasound for 1 h to 8 h at a power of 10 W to 100 W.

6. The preparation method according to any one of claims 1-3, characterized in that, In step S1, the natural limbal tissue is placed in a decellularization solution for decellularization treatment; the decellularization solution comprises the following components: Phospholipase 15ml / L~25ml / L, sodium deoxycholate 8g / L~12g / L, NaCl 5g / L~8g / L, KCl 0.3g / L~0.6g / L, CaCl2 0.1g / L~0.4g / L, MgCl2·6H2O 0.1g / L~0.3g / L, glucose 0.5g / L~1.5g / L, NaHCO3 2g / L~5g / L, antibacterial agent 8ml / L~15ml / L, balance water.

7. The preparation method according to claim 6, characterized in that, The decellularization treatment is performed at a temperature of 35-38°C for 4-12 hours. And / or, the antibacterial agent includes penicillin, streptomycin, and amphotericin B; preferably, the concentration of penicillin is 80 U / ml to 120 U / ml, the concentration of streptomycin is 80 μg / ml to 120 μg / ml, and the concentration of amphotericin B is 0.1 μg / ml to 0.3 μg / ml.

8. The preparation method according to any one of claims 1-3, characterized in that, The natural limbal tissue is xenogeneic, allogeneic, or autologous natural limbal tissue.

9. A decellularized limbal stroma prepared by the method of any one of claims 1-8.

10. The use of the decellularized limbal matrix according to claim 9 in the preparation of limbal epithelial stem cell products.

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