Conjunctival repair material, preparation method therefor and use thereof

Abstract

The present invention relates to the technical field of biomedical materials, and provides a conjunctival repair material, a preparation method therefor and a use thereof. The present invention provides a preparation method for a conjunctival repair material, comprising: carrying out ultrasonic treatment on isolated animal conjunctival tissue; carrying out decellularization treatment on the ultrasonically treated conjunctival tissue under the action of nuclease; and carrying out ultrahigh static pressure crosslinking on the decellularized conjunctival tissue in the presence of a crosslinking agent to obtain the conjunctival repair material, wherein the crosslinking agent comprises the following types of substances: carbodiimide, genipin, epicatechin gallate and proanthocyanidin. The conjunctival repair material prepared by the method of the present invention can improve the mechanical properties of the conjunctival repair material, thereby maintaining the arrangement and integrity of a specific fibrous structure of the conjunctiva, and maintaining the original biological characteristics.

Description

A conjunctival repair material and its preparation method and application

[0001] This application claims priority to the Chinese patent application filed with the China Patent Office on August 24, 2023, with application number 202311068239.8 and invention name “A conjunctival repair material, its preparation method and application”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present invention belongs to the technical field of biomedical materials, and in particular relates to a conjunctival repair material and a preparation method and application thereof. Background Art

[0003] The eyeball is the organ of vision in humans and mammals, and its structure is remarkably delicate. The conjunctiva, located on the surface of the eyeball and in direct contact with the outside world, is susceptible to external damage and endogenous diseases, which can lead to blindness in severe cases. Restoring vision requires rapid and normal regeneration of conjunctival epithelial cells. The mechanism of healing conjunctival damage involves three steps: first, epithelial cell adhesion, expansion, and migration, followed by epithelial cell proliferation, and finally, epithelial cell differentiation. These three steps restore the epithelium to its orderly layered structure.

[0004] Currently, the most common treatment for conjunctival damage is conjunctival reconstruction using normal conjunctiva or conjunctival substitutes, including autologous conjunctival transplantation, lip mucosa transplantation, amniotic membrane transplantation, and allogeneic conjunctival transplantation. However, due to the limited availability of autologous conjunctiva for transplantation and the considerable difficulty in obtaining allogeneic conjunctiva with a good tissue match, the feasibility of treatment is greatly affected.

[0005] Xenotransplantation of conjunctiva utilizes the conjunctiva of an alien animal as material, removing the cells to create a biocompatible tissue structure with morphology and properties similar to those of the human conjunctiva, promoting cell proliferation and function. Decellularized conjunctival tissue requires cross-linking to impart toughness. However, existing cross-linking methods often utilize glutaraldehyde solutions for cross-linking protection. However, glutaraldehyde itself is toxic, and its use in the preparation of biological tissue repair materials inevitably results in residual toxicity. This significantly impacts the clinical application of conjunctival materials.

[0006] Summary of the Invention

[0007] In view of this, the purpose of the present invention is to provide a method for preparing a conjunctival repair material, which adopts ultra-high static pressure cross-linking technology to cross-link the conjunctival tissue, thereby improving the mechanical properties and mechanical properties of the material.

[0008] The present invention provides a method for preparing a conjunctival repair material, comprising the following steps:

[0009] The separated animal conjunctival tissue is ultrasonically treated to obtain ultrasonically treated conjunctival tissue;

[0010] decellularizing the ultrasonically treated conjunctival tissue under the action of nuclease to obtain decellularized conjunctival tissue;

[0011] Decellularized conjunctival tissue is subjected to ultra-high static pressure cross-linking in the presence of a cross-linking agent to obtain a conjunctival repair material; the cross-linking agent includes the following substances: carbodiimide, genipin, epicatechin gallate and proanthocyanidin.

[0012] Preferably, during the cross-linking, the pressure of the ultra-high static pressure treatment is 200 to 800 MPa, and the time of the ultra-high static pressure treatment is 1 to 30 minutes.

[0013] Preferably, the mass concentration of the cross-linking agent is 0.1% to 5%.

[0014] Preferably, the nuclease comprises at least one of DNA enzyme, RNA enzyme, and recombinant nuclease;

[0015] The final concentration of the nuclease is 50 to 1500 U / ml;

[0016] Preferably, the temperature of the decellularization treatment is 20-40° C., and the time of the decellularization treatment is 1-4 hours.

[0017] Preferably, during the decellularization or cross-linking, a protective liquid is used as a medium for treating the material;

[0018] The protective solution is PBS or HBSS buffer containing 5-50 g / L chondroitin sulfate, 5-50 g / L hyaluronic acid, and 5-20 g / L dextran, with a pH value of 6.8-7.4 and an osmotic pressure of 300-400 mOsm.

[0019] Preferably, the frequency of the ultrasonic treatment is 20 to 60 Hz, and the time of the ultrasonic treatment is 2 to 20 minutes.

[0020] Preferably, the animal includes any one of the following: pigs, cattle and sheep.

[0021] The present invention provides a conjunctival repair material prepared by the preparation method, wherein the suture tearing force is not less than 1.0N and the tensile strength is not less than 3.0N.

[0022] The present invention provides application of the conjunctival repair material prepared by the preparation method in preparing engineering materials for repairing conjunctival damage.

[0023] The present invention provides a method for preparing a conjunctival repair material, comprising the following steps: subjecting isolated animal conjunctival tissue to ultrasonic treatment to obtain ultrasonically treated conjunctival tissue; subjecting the ultrasonically treated conjunctival tissue to decellularization under the action of a nuclease to obtain decellularized conjunctival tissue; subjecting the decellularized conjunctival tissue to ultrahigh static pressure cross-linking in the presence of a cross-linking agent to obtain a conjunctival repair material; the cross-linking agent includes the following substances: carbodiimide, genipin, epicatechin gallate, and proanthocyanidins. The present invention first uses ultrasonic oscillation to loosen cells within the conjunctival tissue; utilizing the action of nucleases to quickly and effectively remove cell debris; and utilizing ultrahigh static pressure cross-linking technology to cross-link the conjunctival tissue structure in a relatively short period of time, thereby improving the mechanical and mechanical properties of the conjunctival repair material, thereby maintaining the arrangement and integrity of the conjunctival's unique fiber structure and its original biological properties. Experiments have shown that the conjunctival repair material prepared by the method of the present invention has good tensile strength, which is not less than 3.0N. The conjunctival repair material has good resistance to suture tearing, and the suture tearing force is not less than 1.0N. Compared with the conjunctival repair materials prepared by animal conjunctival tissue and other methods, the tensile strength and suture tearing force are significantly improved.

[0024] Furthermore, the conjunctival repair material prepared using the present invention completely removes foreign cells, well-preserves the extracellular collagen matrix, and maintains the integrity of the original conjunctival tissue structure. α-Gal residue testing also meets requirements, with an α-Gal antigen clearance rate of 99.87%. DNA residue testing also shows an average residual level of 0.88 ng / mg, far below the standard of less than 50 ng / mg for products made from common animal-derived materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] FIG1 is the HE staining result of the decellularized conjunctival tissue material prepared in Example 1, wherein A is the 200-fold magnification observation result of the non-decellularized porcine conjunctival tissue, and B is the 200-fold magnification observation result of the decellularized porcine conjunctival tissue material prepared by the present invention;

[0026] FIG2 is a tensile strength test result of the acellular porcine conjunctival tissue material prepared in Example 1;

[0027] FIG3 is a suture tearing force test result of the acellular porcine conjunctival tissue material prepared in Example 1;

[0028] FIG4 shows the HE staining results of the decellularized conjunctival tissue material prepared in Example 2, wherein A is the 200-fold magnification observation result of the non-decellularized porcine conjunctival tissue, and B is the 200-fold magnification observation result of the decellularized porcine conjunctival tissue material prepared by the present invention;

[0029] FIG5 is a tensile strength test result of the acellular porcine conjunctival tissue material prepared in Example 2;

[0030] FIG6 is a suture tearing force test result of the acellular porcine conjunctival tissue material prepared in Example 2;

[0031] Figure 7 is a morphological diagram of the acellular conjunctival tissue in situ after transplantation in the experimental group of Example 5

[0032] FIG8 is the HE staining results of the acellular conjunctival tissue after transplantation in the experimental group in Example 5;

[0033] Figure 9 is a morphological image of the non-decellularized conjunctival tissue in situ after transplantation in the control group of Example 5

[0034] FIG10 is the HE staining result of the non-decellularized conjunctival tissue after transplantation in the control group in Example 5. DETAILED DESCRIPTION

[0035] The present invention provides a method for preparing a conjunctival repair material, comprising the following steps:

[0036] The separated animal conjunctival tissue is ultrasonically treated to obtain ultrasonically treated conjunctival tissue;

[0037] decellularizing the ultrasonically treated conjunctival tissue under the action of nuclease to obtain decellularized conjunctival tissue;

[0038] Decellularized conjunctival tissue is subjected to ultra-high static pressure cross-linking in the presence of a cross-linking agent to obtain a conjunctival repair material; the cross-linking agent includes the following substances: carbodiimide, genipin, epicatechin gallate and proanthocyanidin.

[0039] The invention performs ultrasonic treatment on the separated animal conjunctival tissue to obtain ultrasonically treated conjunctival tissue.

[0040] In the present invention, the animal preferably includes any one of the following: pigs, cattle and sheep. The present invention has no special restrictions on the method of separating animal conjunctival tissue, and separation methods well known in the art can be used. The frequency of the ultrasonic treatment is preferably 20 to 60 Hz, more preferably 30 to 50 Hz, and most preferably 40 Hz. The time of the ultrasonic treatment is preferably 2 to 20 minutes, more preferably 5 to 15 minutes, and most preferably 10 minutes. Ultrasonic treatment is conducive to loosening the conjunctival tissue, which is conducive to the entry of nucleases into the tissue during subsequent enzymatic hydrolysis of nucleases, thereby improving the enzymatic hydrolysis effect.

[0041] After the ultrasonic treatment is completed, the present invention performs a decellularization treatment on the ultrasonically treated conjunctival tissue under the action of nuclease to obtain decellularized conjunctival tissue.

[0042] In the present invention, the nuclease preferably includes at least one of DNA enzyme, RNA enzyme, and recombinant nuclease. The function of the nuclease is to enzymatically hydrolyze the nucleic acids in the epithelial cells and the lamina propria in the conjunctival tissue. The final concentration of the nuclease is 50 to 1500 U / ml, more preferably 100 to 1000 U / ml, and more preferably 200 U / ml. In an embodiment of the present invention, recombinant nuclease is used for enzymatic hydrolysis. Recombinant nuclease, also known as super nuclease, is purchased from Sino Biological Inc., Beijing, and the product model is SSNP01. The temperature of the decellularization treatment is preferably 20 to 40°C, more preferably 25 to 37°C, and most preferably 30°C. The time of the decellularization treatment is preferably 1 to 4h, more preferably 2 to 3h, and most preferably 2.5h. During the decellularization process, a protective solution is used as the medium for the treatment material. The protective solution is PBS or HBSS buffer containing 5-50 g / L chondroitin sulfate, 5-50 g / L hyaluronic acid, and 5-20 g / L dextran, with a pH of 6.8-7.4 and an osmotic pressure of 300-400 mOsm. More preferably, the protective solution contains 15-25 g / L chondroitin sulfate, 10-15 g / L hyaluronic acid, and 10-15 g / L dextran. The protective solution preferably also includes a detergent. The detergent is at least one of sodium dodecyl sulfate, sodium dodecylbenzenesulfonate, and Triton X-100, used at a concentration of 0.1% to 5%. The detergent can quickly and effectively remove cell debris. The protective solution maintains the colloidal osmotic pressure of the decellularized conjunctiva, significantly reducing excessive swelling of the extracellular matrix during the decellularization process. The decellularization process is preferably accompanied by shaking. The shaking speed is preferably 50-200 rpm, more preferably 100 rpm. The oscillation is conducive to sufficient enzymatic hydrolysis and the freeing of enzymatically hydrolyzed DNA and cell fragments from the tissue, thereby reducing the residual DNA and cell fragments.

[0043] After the decellularization process, the present invention performs ultra-high hydrostatic pressure cross-linking on the decellularized conjunctival tissue in the presence of a cross-linking agent to obtain a conjunctival repair material. The cross-linking agent includes the following substances: carbodiimide, genipin, epicatechin gallate, and proanthocyanidins. The final concentration of the carbodiimide is preferably 0.1%, and the final concentration of genipin or epicatechin gallate is preferably 0.5%.

[0044] In the present invention, during crosslinking, the ultrahigh static pressure treatment pressure is preferably 200-800 MPa, more preferably 300-500 MPa, and most preferably 400 MPa. The ultrahigh static pressure treatment time is preferably 1-30 minutes, more preferably 5-20 minutes, and most preferably 10 minutes. The concentration of the crosslinking agent is preferably 0.5%-1%. During crosslinking, a protective liquid is preferably used as the medium for treating the material; the protective agent formula is the same as above. The use of ultrahigh static pressure crosslinking technology can crosslink conjunctival tissue structures in a relatively short period of time, thereby improving the mechanical and mechanical properties of conjunctival repair materials.

[0045] In the present invention, after the ultrahigh hydrostatic pressure treatment and cross-linking, the cross-linked conjunctival tissue is further cleaned and low-temperature dried. The cleaning solvent is preferably PBS buffer. The cleaning time is preferably 30 minutes. The low-temperature drying temperature is preferably 0-40°C, more preferably 20-35°C, and the drying time is 4-24 hours, more preferably 5 hours.

[0046] The present invention provides a conjunctival repair material prepared by the preparation method, wherein the suture tearing force is not less than 1.0N and the tensile strength is not less than 3.0N.

[0047] The structure of the conjunctival repair material maintains the arrangement and integrity of the unique fiber structure and maintains the original biological properties.

[0048] The present invention provides application of the conjunctival repair material prepared by the preparation method in preparing engineering materials for repairing conjunctival damage.

[0049] In the present invention, the prepared conjunctival repair material is used in an animal model of conjunctival defect. Two weeks after transplantation, the transplanted decellularized conjunctival repair material is in place, the boundary between the conjunctival repair material and the normal conjunctiva basically disappears, and the wound is repaired; while the control material begins to dissolve two weeks after transplantation, accompanied by a certain inflammatory reaction.

[0050] The conjunctival repair material provided by the present invention, its preparation method and application are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0051] Example 1

[0052] A preparation method of porcine conjunctival repair material

[0053] (1) Fresh pig eyeballs were taken, rinsed with physiological saline containing 0.04% tobramycin, fixed on a holder, and the complete conjunctival tissue was cut along the corneoscleral limbus at 10°C and trimmed to a uniform thickness to obtain conjunctival tissue;

[0054] (2) The conjunctival tissue was sealed and subjected to ultrasonic oscillation; the ultrasonic oscillation frequency was 40 Hz and the ultrasonic oscillation time was 2 min;

[0055] (3) The ultrasonically treated conjunctival tissue is sealed in a protective solution for vibration decellularization;

[0056] The protective solution is a PBS buffer containing 50 U / ml of recombinant nuclease, 25 g / L of chondroitin sulfate, 5 g / L of dextran, and 0.02% of Triton X-100. The shaking treatment temperature is 37°C for 2 hours, and then the cells are washed with PBS buffer for 30 minutes (3 times, 10 minutes each time).

[0057] (4) sealing the conjunctival tissue in a crosslinking agent protective solution and crosslinking under ultra-high static pressure conditions;

[0058] The protective solution is a PBS buffer solution containing a crosslinking agent, carbodiimide 0.1%, chondroitin sulfate 25g / L, and dextran 5g / L; the ultra-high static pressure crosslinking condition is 400MPa, and the holding time is 10 minutes;

[0059] (5) The conjunctival tissue was removed, washed with PBS buffer for 30 minutes, flattened, and dried at a constant temperature of 40°C for 4 hours to obtain a bioactive acellular conjunctival repair material.

[0060] Example 2

[0061] A preparation method of porcine conjunctival repair material

[0062] (1) Fresh pig eyeballs were taken, rinsed with physiological saline containing 0.1% tobramycin, fixed on a holder, and the complete conjunctival tissue was cut along the corneoscleral limbus at 4°C and trimmed to a uniform thickness to obtain conjunctival tissue;

[0063] (2) The conjunctival tissue was sealed and subjected to ultrasonic oscillation; the ultrasonic oscillation frequency was 20 Hz and the ultrasonic oscillation time was 5 min;

[0064] (3) The ultrasonically treated conjunctival tissue is sealed in a protective solution for vibration decellularization;

[0065] The protective solution is a PBS buffer solution containing 100 U / ml of recombinant nuclease, 5 g / L of chondroitin sulfate, 15 g / L of hyaluronic acid, 10 g / L of dextran, and 0.2% of sodium dodecylbenzenesulfonate. The shaking treatment temperature is 26°C, the treatment time is 4 hours, and then the cells are washed with PBS buffer solution for 30 minutes (3 times, each time for 10 minutes);

[0066] (4) sealing the conjunctival tissue in a crosslinking agent protective solution and crosslinking under ultra-high static pressure conditions;

[0067] The protective solution is a PBS buffer solution containing a cross-linking agent genipin 0.5%, chondroitin sulfate 5g / L, hyaluronic acid 15g / L, and dextran 10g / L; the ultra-high static pressure cross-linking condition is 600MPa, and the holding time is 5 minutes;

[0068] (5) The conjunctival tissue was removed, washed with PBS buffer for 30 minutes, flattened, and dried at a constant temperature of 40°C for 4 hours to obtain a bioactive acellular conjunctival repair material.

[0069] Example 3

[0070] A preparation method of bovine conjunctival repair material

[0071] (1) Fresh bovine eyeballs were rinsed with physiological saline containing 0.05% tobramycin, fixed on a holder, and the complete conjunctival tissue was cut along the corneoscleral limbus at 4°C and trimmed to a uniform thickness to obtain conjunctival tissue;

[0072] (2) The conjunctival tissue was sealed and subjected to ultrasonic oscillation; the ultrasonic oscillation frequency was 40 Hz and the ultrasonic oscillation time was 2 min;

[0073] (3) The ultrasonically treated conjunctival tissue is sealed in a protective solution for vibration decellularization;

[0074] The protective solution is a PBS buffer solution containing 200 U / ml of recombinant nuclease, 15 g / L of chondroitin sulfate, 10 g / L of hyaluronic acid, 5 g / L of dextran, and 0.2% of sodium lauryl sulfate. The shaking treatment temperature is 30°C, the treatment time is 2 hours, and then the cells are washed with PBS buffer solution for 30 minutes (3 times, each time for 10 minutes);

[0075] (4) sealing the conjunctival tissue in a crosslinking agent protective solution and crosslinking under ultra-high static pressure conditions;

[0076] The protective solution is a PBS buffer solution containing a cross-linking agent, epicatechin gallate 0.5%, chondroitin sulfate 15g / L, hyaluronic acid 10g / L, and dextran 5g / L; the ultra-high static pressure cross-linking condition is 800MPa, and the holding time is 5 minutes;

[0077] (5) The conjunctival tissue was removed, washed with PBS buffer for 30 minutes, flattened, and dried at a constant temperature of 40°C for 4 hours to obtain a bioactive acellular conjunctival repair material.

[0078] Example 4

[0079] Residue detection of bioactive conjunctival repair materials

[0080] (1) HE staining experiment: The decellularized conjunctival repair material obtained in Example 1 and the non-decellularized porcine conjunctival tissue were stained with HE according to conventional methods.

[0081] The results are shown in Figure 1. The xenogeneic cells in the acellular conjunctival repair material were completely removed, the extracellular collagen fiber matrix structure was retained, and the original tissue structure of the conjunctiva remained intact.

[0082] (2) Residual α-Gal Residue Detection: Referring to the method in the industry standard "Residual α-Gal Antigen Detection of Animal-derived Scaffold Materials for Tissue Engineering Medical Devices" (YY / T 1561-2017), the α-Gal antigen content of porcine conjunctival tissue and the decellularized conjunctival repair material obtained in Example 1 was determined. The main experimental process included: sample homogenization and lysate digestion, sample reaction with specific anti-α-Gal antibodies, and ELISA detection of residual antibodies using a-Gal antigen quantitative detection kit (purchased from Beijing Sanyao Technology Development Co., Ltd., product number: 70101). α-Gal antigen content determination results (see Table 1): Porcine conjunctival α-Gal antigen content: (1.50±0.15)×10 16 / mg (dry weight). α-Gal antigen content of acellular conjunctival repair material: (1.85±0.09)×10 13 The α-Gal antigen clearance rate of the acellular conjunctival repair material was 99.87%.

[0083] Table 1 Detection results of α-Gal antigen content in samples *: (number of α-Gal antigen epitopes in porcine conjunctiva - number of α-Gal antigen epitopes in acellular conjunctival repair material) / number of α-Gal antigen epitopes in porcine conjunctiva × 100.

[0084] (3) DNA residue detection: According to the method of YY / T 0606.25-2014 "Tissue Engineering Medical Products Part 25: Determination of DNA Residue in Animal-derived Biomaterials: Fluorescence Staining Method", the DNA residue was determined on the porcine conjunctival tissue and the decellularized conjunctival repair material obtained in Example 1. The main experimental process includes: sample preparation and enzymatic digestion, DNA purification, application of double-stranded DNA fluorescent dye to specifically bind to double-stranded DNA to form a complex, and use of a fluorescence microplate reader to generate an ultra-strong fluorescence signal under excitation at a wavelength of 480 nm, with 520 nm as the emission wavelength and 530 nm as the cutoff wavelength for detection. Within a certain DNA concentration range and when the fluorescent dye is in excess, the fluorescence intensity is proportional to the DNA concentration. The DNA residue in the test sample is calculated based on the fluorescence intensity of the test sample. Three parallel samples are set.

[0085] DNA residue test results: Sample 1 was 0.87ng / mg, Sample 2 was 0.96ng / mg, and Sample 3 was 0.81ng / mg. The average was 0.88ng / mg, which is far lower than the standard requirement of less than 50ng / mg for DNA residue in products made from common animal-derived materials.

[0086] (4) Tensile strength and suture tearing force testing

[0087] 1) Tensile strength test: Take the porcine conjunctival tissue, the non-cross-linked decellularized conjunctival repair material and the decellularized conjunctival repair material obtained in Example 1, soak them in physiological saline for 1 minute, take them out, cut them into a rectangle of 3mm×7mm, and use an electronic tensile testing machine to measure the tensile strength of the samples. The two sides of the sample are clamped and fixed on the clamp (the longitudinal axis of the sample coincides with the line connecting the centers of the upper and lower clamps, and the tightness is appropriate). Stretch at a steady speed of 50mm / min until it breaks, and record the maximum load force when the sample breaks. At the same time, porcine conjunctival tissue (without any treatment) and non-decellularized porcine conjunctival tissue material are set (the preparation method is described in Example 1 of CN105770994A patent). The maximum load forces of the three groups of samples of porcine conjunctival tissue, non-decellularized porcine conjunctival tissue material and decellularized conjunctival repair material prepared by the present invention are 2.2N, 2.1N and 3.5N respectively.

[0088] The test results show that the tensile strength of the samples prepared by this method is significantly improved (see Figure 2).

[0089] 2) Suture tearing force test: Take pig eye conjunctival tissue, non-cross-linked decellularized conjunctival repair material and the decellularized conjunctival repair material obtained in Example 1, soak them in physiological saline for 1 minute, take them out, cut them into 5mm×5mm squares, and pass them through the center point of the product with 10-0 nylon thread. Use an electronic tensile testing machine to measure the sample suture tearing force. Fix one end of the sample to the clamp, and fix the tail end of the suture with the clamp after it hangs freely to ensure that the experimental sample is not stretched or damaged by the clamp. The suture is stretched at a steady speed of 50mm / min, and the pulling force that pulls the suture out of the sample or damages the sample is recorded. At the same time, pig conjunctival tissue (without any treatment) and non-decellularized pig conjunctival tissue material (preparation method is described in Example 1 of CN105770994A patent) are set. At the same time, pig conjunctival tissue (without any treatment) and non-decellularized pig conjunctival tissue material (preparation method is described in Example 1 of CN105770994A patent) are set. The tensile forces of the three groups of samples, namely, porcine conjunctival tissue, non-decellularized porcine conjunctival tissue material, and the decellularized conjunctival repair material prepared by the present invention, were 0.69N, 0.72N, and 1.04N, respectively.

[0090] The test results show that the tearing strength of the sample suture prepared by this method is significantly improved (see Figure 3).

[0091] Example 5

[0092] Detection of the acellular conjunctival repair material prepared in Example 2

[0093] (1) HE staining experiment: The decellularized conjunctival repair material obtained in Example 2 and the non-decellularized porcine conjunctival tissue were subjected to HE staining according to conventional methods. The results are shown in FIG4 . The heterologous cells in the decellularized conjunctival repair material were completely removed, the extracellular collagen fiber matrix structure was retained, and the original tissue structure of the conjunctiva remained intact.

[0094] (3) DNA residue detection: According to the method of YY / T 0606.25-2014 "Tissue Engineering Medical Products Part 25: Determination of DNA Residue in Animal-Derived Biomaterials: Fluorescence Staining Method", the DNA residue in porcine conjunctival tissue and the decellularized conjunctival repair material obtained in Example 1 were determined. The main experimental process includes: sample preparation and enzymatic digestion, DNA purification, application of double-stranded DNA fluorescent dye to specifically bind to double-stranded DNA to form a complex, and detection using a fluorescence microplate reader at a wavelength of 480nm to generate a super-strong fluorescence signal, 520nm as the emission wavelength, and 530nm as the cutoff wavelength. Within a certain DNA concentration range and when the fluorescent dye is in excess, the fluorescence intensity is proportional to the DNA concentration. The DNA residue in the test sample is calculated based on the fluorescence intensity of the test sample. The results of DNA residue detection are: 0.57ng / mg for sample 1, 0.55ng / mg for sample 2, and 0.53ng / mg for sample 3. The average is 0.55ng / mg, which is far lower than the standard requirement that the DNA residue of products made from common animal-derived materials is less than 50ng / mg.

[0095] (4) Tensile strength and suture tearing force testing

[0096] 1) Tensile strength test: Take the porcine conjunctival tissue and the decellularized conjunctival repair material obtained in Example 2, soak them in physiological saline for 1 minute, take them out, cut them into a rectangle of 3mm×7mm, and use an electronic tensile testing machine to measure the tensile strength of the samples. The two sides of the sample are clamped and fixed on the clamp (the longitudinal axis of the sample coincides with the line connecting the centers of the upper and lower clamps, and the tightness is appropriate). Stretch at a steady speed of 50mm / min until it breaks, and record the maximum load force when the sample breaks. At the same time, set up porcine conjunctival tissue (without any treatment) and non-decellularized porcine conjunctival tissue material (the preparation method is described in Example 1 of CN105770994A patent). The maximum load forces of the three groups of samples of porcine conjunctival tissue, non-decellularized porcine conjunctival tissue material and decellularized conjunctival repair material prepared by the present invention are 2.5N, 2.5N and 3.6N, respectively. The test results show that the tensile strength of the samples prepared by this method is significantly improved (see Figure 5).

[0097] 2) Suture tearing force test: Take the conjunctival tissue of the pig eye and the decellularized conjunctival repair material obtained in Example 2, soak them in physiological saline for 1 minute, take them out, cut them into 5mm×5mm squares, and pass them through the center point of the product with 10-0 nylon thread. Use an electronic tensile testing machine to measure the tearing force of the sample sutures. Fix one end of the sample to the clamp, and fix the tail end of the suture with the clamp after it hangs freely to ensure that the experimental sample is not stretched or damaged by the clamp. The suture is stretched at a steady speed of 50mm / min, and the pulling force that pulls the suture out of the sample or damages the sample is recorded. At the same time, pig conjunctival tissue (without any treatment) and non-decellularized pig conjunctival tissue material (preparation method is described in Example 1 of CN105770994A patent). The pulling forces of the three groups of samples of pig conjunctival tissue, non-decellularized pig conjunctival tissue material and decellularized conjunctival repair material prepared by the present invention are 0.73N, 0.78N and 1.22N respectively. The test results show that the tearing strength of the sample suture prepared by this method is significantly improved (see Figure 6).

[0098] Example 5

[0099] Experimental results of the animal model of the acellular conjunctival repair material prepared in Example 1

[0100] A conjunctival defect model was constructed by removing a portion of the bulbar conjunctiva (approximately 10 mm × 6 mm) from the ocular surface of healthy New Zealand white rabbits, located to the left of the right rectus muscle. The rabbits were randomly divided into an experimental group and a control group. The experimental group received a transplant of the acellular conjunctival tissue obtained in Example 1 at the defect site, while the control group received a transplant of a biological amniotic membrane at the defect site.

[0101] Two weeks after transplantation, the bulbar conjunctiva on the left side of the right rectus muscle of the rabbits in the two groups was photographed, and the repaired conjunctival tissue sections were stained with HE method and observed.

[0102] The results showed that after 2 weeks, the acellular conjunctival repair material was in place in the experimental group, the boundary between the conjunctival repair material and the normal conjunctiva basically disappeared, and the wound surface was repaired (see Figures 7 and 8). After 2 weeks, the conjunctival repair material in the control group began to dissolve, and the conjunctival tissue was observed to be severely congested, and HE staining showed an increase in purple-red inflammatory cells, indicating that a certain inflammatory response occurred (see Figures 9 and 10).

[0103] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for preparing a conjunctival repair material, characterized in that: The following steps are involved: The separated animal conjunctival tissue is subjected to ultrasonic treatment to obtain ultrasonic treated conjunctival tissue; Decellularizing the ultrasonically treated conjunctival tissue under the action of nuclease to obtain decellularized conjunctival tissue; The decellularized conjunctival tissue is subjected to ultra-high static pressure treatment and cross-linking in the presence of a cross-linking agent to obtain a conjunctival repair material; the cross-linking agent includes any of the following substances: carbodiimide, genipin, epicatechin gallate and proanthocyanidin.

2. The preparation method according to claim 1, characterized in that: During the cross-linking, the pressure of the ultra-high static pressure treatment is 200-800 MPa, and the time of the ultra-high static pressure treatment is 1-30 minutes.

3. The preparation method according to claim 2, characterized in that: During the cross-linking, the pressure of the ultra-high static pressure treatment is 300-500 MPa, and the time of the ultra-high static pressure treatment is 5-20 minutes.

4. The preparation method according to claim 1, characterized in that: The mass concentration of the cross-linking agent is 0.1% to 5%.

5. The preparation method according to claim 1, characterized in that: The nuclease includes at least one of DNA enzyme, RNA enzyme and recombinant nuclease.

6. The preparation method according to claim 1, characterized in that: The final concentration of the nuclease is 50-1500 U / ml.

7. The preparation method according to claim 6, characterized in that: The final concentration of the nuclease is 100-200 U / ml.

8. The preparation method according to claim 1, characterized in that: The temperature of the decellularization treatment is 20 to 40° C., and the time of the decellularization treatment is 1 to 4 hours.

9. The preparation method according to claim 1, characterized in that: During the decellularization or cross-linking, a protective liquid is used as a medium for treating the material; The protective solution is PBS or HBSS buffer solution containing 5-50 g / L chondroitin sulfate, 5-50 g / L hyaluronic acid and 5-20 g / L dextran, with a pH value of 6.8-7.4 and an osmotic pressure of 300-400 mOsm.

10. The preparation method according to claim 9, characterized in that: During the decellularization treatment, the protective solution further comprises a detergent; The detergent is at least one of sodium dodecyl sulfate, sodium dodecylbenzene sulfonate and Triton X-100.

11. The preparation method according to claim 1, characterized in that: The frequency of the ultrasonic treatment is 20 to 60 Hz, and the time of the ultrasonic treatment is 2 to 20 minutes.

12. The preparation method according to any one of claims 1 to 11, characterized in that: The animal includes any one of the following: pigs, cattle and sheep.

13. The conjunctival repair material prepared by the preparation method according to any one of claims 1 to 12, characterized in that: The tearing force of the suture shall not be less than 1.0N; the tensile strength shall not be less than 3.0N.

14. Use of the conjunctival repair material according to claim 13 in preparing engineering materials for repairing conjunctival damage.

15. Use of the conjunctival repair material according to claim 13 in treating conjunctival damage.