A conjunctival repair material, its preparation method and application

Abstract

The present invention provides a conjunctival repair material, a preparation method thereof and an application, belonging to the technical field of biomedical materials. The present invention provides a preparation method of a conjunctival repair material, which comprises subjecting the separated animal conjunctival tissue to ultrasonic treatment, subjecting the ultrasonically treated conjunctival tissue to decellularization treatment under the action of nuclease, and subjecting the decellularized conjunctival tissue to ultra-high hydrostatic pressure treatment and crosslinking in the presence of a crosslinking agent to obtain a conjunctival repair material; the crosslinking agent includes the following substances: carbodiimide, genipin, epigallocatechin gallate and proanthocyanidin. The conjunctival repair material prepared by the above method of the present invention can improve the mechanical properties and mechanical performance of the conjunctival repair material, so as to maintain the arrangement, integrity and original biological characteristics of the specific fiber structure of the conjunctiva.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and particularly relates to a conjunctival repair material, a preparation method thereof, and an application thereof. Background Art

[0002] The eyeball is the visual organ of humans and mammals, and the structure of the eyeball is very delicate. The conjunctiva is on the surface of the eyeball, directly in contact with the outside world, and is very vulnerable to external injuries. At the same time, endogenous diseases are also likely to occur, and in severe cases, blindness may result. Restoring vision requires the rapid and normal regeneration of conjunctival epithelial cells to cover. The mechanism of curing conjunctival injury includes the following three steps: First, the adhesion, stretching, and migration of epithelial cells, then the proliferation of epithelial cells, and finally the differentiation of epithelial cells. Only after these three steps can the epithelium return to an orderly arranged layered structure.

[0003] Currently, the commonly used treatment method for conjunctival injury is to reconstruct the conjunctiva with normal conjunctiva or conjunctival substitutes, including autologous conjunctival transplantation, labial mucosa transplantation, amniotic membrane transplantation, allogeneic conjunctival transplantation, etc. However, due to the limited availability of autologous conjunctiva for transplantation, it is quite difficult to obtain allogeneic conjunctiva with good tissue typing, which greatly affects the feasibility of treatment.

[0004] Xenogeneic conjunctival transplantation uses heterologous animal conjunctiva as a material. After removing cells, a biocompatible material with a tissue structure and performance similar to that of human conjunctiva is prepared, which is beneficial to the proliferation and function of cells. For the decellularized conjunctival tissue, cross-linking treatment is required to make the tissue material have toughness. However, in the prior art, the commonly used material for cross-linking is glutaraldehyde solution for cross-linking protection. However, glutaraldehyde reagent itself has certain toxicity, and its application in the preparation of biological tissue repair materials will inevitably lead to the residue of the material. This greatly affects the clinical application of conjunctival materials. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a preparation method of a conjunctival repair material, which uses ultra-high hydrostatic pressure cross-linking technology to cross-link conjunctival tissue, which is beneficial to improving the mechanical properties and mechanical performance of the material.

[0006] The present invention provides a preparation method of a conjunctival repair material, comprising the following steps:

[0007] Subject the separated animal conjunctival tissue to ultrasonic treatment to obtain ultrasonically treated conjunctival tissue;

[0008] Subject the ultrasonically treated conjunctival tissue to decellularization treatment under the action of nuclease to obtain decellularized conjunctival tissue;

[0009] The acellular conjunctival tissue is crosslinked by ultra-high hydrostatic pressure treatment in the presence of a crosslinking agent to obtain a conjunctival repair material; the crosslinking agent includes any one of the following substances: carbodiimide, genipin, epigallocatechin gallate, and procyanidin.

[0010] Preferably, during the crosslinking, the pressure of the ultra-high hydrostatic pressure treatment is 200 - 800 MPa, and the time of the ultra-high hydrostatic pressure treatment is 1 - 30 min.

[0011] Preferably, the mass concentration of the crosslinking agent is 0.1% - 5%.

[0012] Preferably, the nuclease includes at least one of DNase, RNase, and recombinant nuclease;

[0013] The final concentration of the nuclease is 50 - 1500 U / ml.

[0014] Preferably, the temperature of the acellular treatment is 20 - 40 °C, and the time of the acellular treatment is 1 - 4 h.

[0015] Preferably, during the acellular treatment or crosslinking, a protective solution is used as the medium for the treatment material;

[0016] The protective solution is a PBS or HBSS buffer containing 5 - 50 g / L of chondroitin sulfate, 5 - 50 g / L of hyaluronic acid, and 5 - 20 g / L of dextran, with a pH value of 6.8 - 7.4 and an osmotic pressure of 300 - 400 mOsm.

[0017] Preferably, the frequency of the ultrasonic treatment is 20 - 60 Hz, and the time of the ultrasonic treatment is 2 - 20 min.

[0018] Preferably, the animal includes any one of the following: pig, cow, and sheep.

[0019] The present invention provides a conjunctival repair material prepared by the preparation method, with a suture tearing force of not less than 1.0 N; the tensile strength is not less than 3.0 N.

[0020] The present invention provides the application of the conjunctival repair material prepared by the preparation method in the preparation of engineering materials for conjunctival injury repair.

[0021] The present invention provides a method for preparing a conjunctival repair material, comprising the following steps: subjecting the isolated animal conjunctival tissue to ultrasonic treatment to obtain ultrasonically treated conjunctival tissue; subjecting the ultrasonically treated conjunctival tissue to decellularization treatment under the action of nuclease to obtain decellularized conjunctival tissue; subjecting the decellularized conjunctival tissue to ultra-high hydrostatic pressure treatment and crosslinking in the presence of a crosslinking agent to obtain a conjunctival repair material; the crosslinking agent includes any one of the following substances: carbodiimide, genipin, epigallocatechin gallate, and procyanidin. The present invention first uses ultrasonic oscillation to loosen the cells in the conjunctival tissue; by the action of detergents and nucleases, cell debris can be quickly and effectively removed; the ultra-high hydrostatic pressure crosslinking technology can crosslink the conjunctival tissue structure in a short time, improving the mechanical properties and mechanical performance of the conjunctival repair material, thereby maintaining the arrangement, integrity, and original biological characteristics of the unique fiber structure of the conjunctiva. Experiments have proved that the conjunctival repair material prepared by the preparation method of the present invention has good tensile strength, and the tensile strength is not less than 3.0 N. The conjunctival repair material has good ability to resist suture tearing, and the suture tearing force is not less than 1.0 N. Compared with animal conjunctival tissue and conjunctival repair materials prepared by other methods, the tensile strength and suture tearing force are significantly improved.

[0022] At the same time, for the conjunctival repair material prepared by the preparation method of the present invention, xenogeneic cells are completely removed, the extracellular collagen fiber matrix structure is well preserved, and the original tissue structure of the conjunctiva remains intact. And the detection of α-Gal residue meets the requirements, the clearance rate of α-Gal antigen is 99.87%. The DNA residue detection shows that the average residual amount of the DNA residue detection result is 0.88 ng / mg, which is far less than the standard requirement that the DNA residual amount of products made of common animal-derived materials is less than 50 ng / mg. Description of the Drawings

[0023] Figure 1 The HE staining results of the decellularized conjunctival tissue material prepared in Example 1, where A is the observation result of the non-decellularized porcine eye conjunctival tissue at 200 times magnification, and B is the observation result of the decellularized porcine eye conjunctival tissue material prepared by the present invention at 200 times magnification;

[0024] Figure 2 The tensile strength test results of the decellularized porcine eye conjunctival tissue material prepared in Example 1;

[0025] Figure 3 The suture tearing force test results of the decellularized porcine eye conjunctival tissue material prepared in Example 1;

[0026] Figure 4The HE staining results of the acellular conjunctival tissue material prepared in Example 2, where A is the observation result of the non - acellular porcine eye conjunctival tissue under 200 - fold magnification, and B is the observation result of the acellular porcine eye conjunctival tissue material prepared by the present invention under 200 - fold magnification;

[0027] Figure 5 The tensile strength test results of the acellular porcine eye conjunctival tissue material prepared in Example 2;

[0028] Figure 6 The suture tearing force test results of the acellular porcine eye conjunctival tissue material prepared in Example 2;

[0029] Figure 7 The in - situ morphology diagram of the transplanted acellular conjunctival tissue in the experimental group of Example 5

[0030] Figure 8 The HE staining results of the transplanted acellular conjunctival tissue in the experimental group of Example 5;

[0031] Figure 9 The in - situ morphology diagram of the transplanted non - acellular control conjunctival tissue in the control group of Example 6

[0032] Figure 10 The HE staining results of the transplanted non - acellular control conjunctival tissue in the control group of Example 6. Detailed implementation manners

[0033] The present invention provides a preparation method of a conjunctival repair material, comprising the following steps:

[0034] Ultrasonically process the isolated animal conjunctival tissue to obtain ultrasonically processed conjunctival tissue;

[0035] Perform acellular treatment on the ultrasonically processed conjunctival tissue under the action of nuclease to obtain acellular conjunctival tissue;

[0036] Perform ultra - high hydrostatic pressure treatment and cross - linking on the acellular conjunctival tissue in the presence of a cross - linker to obtain a conjunctival repair material; the cross - linker includes any one of the following substances: carbodiimide, genipin, epigallocatechin gallate, and procyanidin.

[0037] The present invention ultrasonically processes the isolated animal conjunctival tissue to obtain ultrasonically processed conjunctival tissue.

[0038] In the present invention, the animal preferably includes any one of the following: pigs, cows, and sheep. The present invention has no special limitation on the method for separating animal conjunctival tissue, and the well-known separation methods in the art can be adopted. The frequency of the ultrasonic treatment is preferably 20 - 60 Hz, more preferably 30 - 50 Hz, and most preferably 40 Hz. The time of the ultrasonic treatment is preferably 2 - 20 min, more preferably 5 - 15 min, and most preferably 10 min. Ultrasonic treatment is beneficial to making the conjunctival tissue loose, which is conducive to the nuclease entering the tissue during subsequent enzymatic hydrolysis and improving the enzymatic hydrolysis effect.

[0039] After the ultrasonic treatment ends, in the present invention, the conjunctival tissue treated by ultrasonic is subjected to decellularization treatment under the action of nuclease to obtain decellularized conjunctival tissue.

[0040] In the present invention, the nuclease preferably includes at least one of DNase, RNase, and recombinant nuclease. The function of the nuclease is to enzymatically hydrolyze the nucleic acids in the epithelial cells and the lamina propria of the conjunctival tissue. The final concentration of the nuclease is 50 - 1500 U / ml, more preferably 100 - 1000 U / ml, and more preferably 200 U / ml. In the embodiments of the present invention, recombinant nuclease is used for enzymatic hydrolysis. Recombinant nuclease, also known as super nuclease, is purchased from SinoBiological Inc., and the product model is SSNP01. The temperature of the decellularization treatment is preferably 20 - 40 °C, more preferably 25 - 37 °C, and most preferably 30 °C. The time of the decellularization treatment is preferably 1 - 4 h, more preferably 2 - 3 h, and most preferably 2.5 h. During the decellularization treatment, a protective solution is used as the medium for the treatment material; the protective solution is a PBS or HBSS buffer solution containing 5 - 50 g / L of chondroitin sulfate, 5 - 50 g / L of hyaluronic acid, and 5 - 20 g / L of dextran, with a pH value of 6.8 - 7.4 and an osmotic pressure of 300 - 400 mOsm; more preferably, it is a PBS or HBSS buffer solution containing 15 - 25 g / L of chondroitin sulfate, 10 - 15 g / L of hyaluronic acid, and 10 - 15 g / L of dextran. The protective solution preferably further includes a detergent. The detergent is at least one of sodium dodecyl sulfate, sodium dodecylbenzenesulfonate, and Triton X - 100, and the use concentration is 0.1% - 5%. The detergent can quickly and effectively remove cell debris. The protective solution maintains the colloid osmotic pressure of the decellularized conjunctiva and can significantly reduce the excessive swelling of the extracellular matrix during decellularization. The decellularization treatment is preferably accompanied by oscillation. The rotation speed of the oscillation is preferably 50 - 200 rpm, more preferably 100 rpm. The oscillation is beneficial to sufficient enzymatic hydrolysis and the free release of the enzymatically hydrolyzed DNA and cell debris from the tissue, thereby reducing the residue of DNA and cell debris.

[0041] After the decellularization process, the present invention subjects the decellularized conjunctival tissue to ultra-high hydrostatic pressure treatment for crosslinking in the presence of a crosslinking agent to obtain a conjunctival repair material; the crosslinking agent includes substances of the following types: carbodiimide, genipin, epigallocatechin gallate, and procyanidin.

[0042] In the present invention, during the crosslinking, the pressure of the ultra-high hydrostatic pressure treatment is preferably 200 - 800 MPa, more preferably 300 - 500 MPa, and most preferably 400 MPa. The time of the ultra-high hydrostatic pressure treatment is preferably 1 - 30 min, more preferably 5 - 20 min, and most preferably 10 min. The concentration of the crosslinking agent is preferably 0.5% - 1%. During the crosslinking, it is preferred to use a protective solution as the medium for the treatment material; the formulation of the protective agent is the same as above. Using the ultra-high hydrostatic pressure crosslinking technology can crosslink the conjunctival tissue structure in a short time, improving the mechanical properties and mechanical performance of the conjunctival repair material.

[0043] In the present invention, after the ultra-high hydrostatic pressure treatment for crosslinking, it further includes washing and low-temperature drying of the crosslinked conjunctival tissue. The solvent for washing is preferably PBS buffer solution. The time for washing is preferably 30 min. The temperature for low-temperature drying is preferably 0 - 40 °C, more preferably 20 - 35 °C; the time is 4 - 24 h, more preferably 5 h.

[0044] The present invention provides a conjunctival repair material prepared by the above preparation method, with a suture tearing force of not less than 1.0 N; a tensile strength of not less than 3.0 N.

[0045] The structure of the conjunctival repair material maintains the arrangement, integrity, and original biological characteristics of the specific fiber structure.

[0046] The present invention provides the application of the conjunctival repair material prepared by the above preparation method in the preparation of engineering materials for conjunctival injury repair.

[0047] In the present invention, when the prepared conjunctival repair material is used in a conjunctival defect animal model, after 2 weeks of transplantation, the transplanted decellularized conjunctival repair material is in place, and the boundary between the conjunctival repair material and the normal conjunctiva basically disappears, and the wound is repaired; while the control material starts to dissolve after 2 weeks of transplantation, and there is a certain inflammatory reaction.

[0048] The following is a detailed description of a conjunctival repair material and its preparation method and application provided by the present invention in conjunction with examples, but they cannot be understood as limiting the protection scope of the present invention.

[0049] Example 1

[0050] A preparation method of a porcine conjunctival repair material

[0051] (1) Take fresh porcine eyeballs, rinse them with physiological saline containing 0.04% tobramycin, fix them on a fixator, and cut the intact conjunctival tissue along the limbus at 10 °C, and trim it to a uniform thickness to obtain conjunctival tissue;

[0052] (2) Seal the conjunctival tissue and perform ultrasonic oscillation; the ultrasonic oscillation frequency is 40 Hz, and the ultrasonic oscillation time is 2 min;

[0053] (3) Seal the ultrasonically treated conjunctival tissue in a protective solution and perform oscillatory decellularization treatment;

[0054] The protective solution is a PBS buffer solution 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 oscillatory treatment temperature is 37 °C, the treatment time is 2 hours, and then wash it with PBS buffer solution by oscillation for 30 minutes (3 times, 10 minutes each time);

[0055] (4) Seal the conjunctival tissue in a cross-linking agent protective solution and perform cross-linking under ultra-high hydrostatic pressure conditions;

[0056] The protective solution is a PBS buffer solution containing 0.1% of cross-linking agent carbodiimide, 25 g / L of chondroitin sulfate, and 5 g / L of dextran; the ultra-high hydrostatic pressure cross-linking conditions are 400 MPa, and the holding time is 10 minutes;

[0057] (5) Take out the conjunctival tissue, wash it with PBS buffer solution by oscillation for 30 minutes, flatten it, and dry it at a constant temperature of 40 °C for 4 hours to obtain a bioactive acellular conjunctival repair material.

[0058] Example 2

[0059] A preparation method of a porcine conjunctival repair material

[0060] (1) Take fresh porcine eyeballs, rinse them with physiological saline containing 0.1% tobramycin, fix them on a fixator, and cut the intact conjunctival tissue along the limbus at 4 °C, and trim it to a uniform thickness to obtain conjunctival tissue;

[0061] (2) Seal the conjunctival tissue and perform ultrasonic oscillation; the ultrasonic oscillation frequency is 20 Hz, and the ultrasonic oscillation time is 5 min;

[0062] (3) Seal the ultrasonically treated conjunctival tissue in a protective solution and perform oscillatory decellularization treatment;

[0063] 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 oscillation treatment temperature is 26°C, the treatment time is 4 hours, and then it is washed by oscillation with PBS buffer solution for 30 minutes (3 times, 10 minutes each time);

[0064] (4) Seal the conjunctival tissue in the crosslinking agent protective solution and perform crosslinking under ultra-high hydrostatic pressure;

[0065] The protective solution is a PBS buffer solution containing 0.5% of the crosslinking agent genipin, 5 g / L of chondroitin sulfate, 15 g / L of hyaluronic acid, and 10 g / L of dextran; the ultra-high hydrostatic pressure crosslinking conditions are 600 MPa, and the holding time is 5 minutes;

[0066] (5) Take out the conjunctival tissue, wash it by oscillation with PBS buffer solution for 30 minutes, flatten it, and dry it at a constant temperature of 40°C for 4 hours to obtain a bioactive acellular conjunctival repair material.

[0067] Example 3

[0068] A preparation method of a bovine conjunctival repair material

[0069] (1) Take a fresh bovine eyeball, rinse it with physiological saline containing 0.05% tobramycin, fix it on a fixator, and cut the complete conjunctival tissue along the limbus corneae at 4°C, and trim it to a uniform thickness to obtain conjunctival tissue;

[0070] (2) Seal the conjunctival tissue and perform ultrasonic oscillation; the ultrasonic oscillation frequency is 40 Hz, and the ultrasonic oscillation time is 2 min;

[0071] (3) Seal the conjunctival tissue after ultrasonic treatment in a protective solution and perform oscillatory decellularization treatment;

[0072] 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 dodecyl sulfate. The oscillation treatment temperature is 30°C, the treatment time is 2 hours, and then it is washed by oscillation with PBS buffer solution for 30 minutes (3 times, 10 minutes each time);

[0073] (4) Seal the conjunctival tissue in the crosslinking agent protective solution and perform crosslinking under ultra-high hydrostatic pressure;

[0074] The protective solution is a PBS buffer solution containing 0.5% of the crosslinking agent epigallocatechin gallate, 15 g / L of chondroitin sulfate, 10 g / L of hyaluronic acid, and 5 g / L of dextran; the ultra-high hydrostatic pressure crosslinking conditions are 800 MPa, and the holding time is 5 minutes;

[0075] (5) Take out the conjunctival tissue, wash it by shaking with PBS buffer for 30 minutes, flatten it, and dry it at a constant temperature of 40 °C for 4 hours to obtain a bioactive acellular conjunctival repair material.

[0076] Example 4

[0077] Residual detection of bioactive conjunctival repair material

[0078] (1) HE staining experiment: The acellular conjunctival repair material obtained in Example 1 and the non-acellular porcine eye conjunctival tissue were stained by HE according to the conventional method.

[0079] The results are shown in Figure 1 As shown, 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.

[0080] (2) Residual α-Gal detection: Referring to the method in the industry standard "Detection of Residual α-Gal Antigen in Animal-derived Scaffold Materials for Tissue Engineering Medical Devices" (YY / T 1561-2017), the α-Gal antigen content of porcine eye conjunctival tissue and the acellular conjunctival repair material obtained in Example 1 was determined. The main experimental procedures included: sample homogenization treatment and digestion with lysis solution, reaction of the sample with specific anti-α-Gal antibody, and detection of the remaining antibody by ELISA using an α-Gal antigen quantitative detection kit (purchased from Beijing Sanyao Science and Technology Development Company, product number: 70101). The results of α-Gal antigen content determination (see Table 1): α-Gal antigen content in porcine eye conjunctiva: (1.50 ± 0.15) × 10 16 cells / mg (dry weight). α-Gal antigen content in the acellular conjunctival repair material: (1.85 ± 0.09) × 10 13 cells / mg (dry weight). The α-Gal antigen clearance rate of the acellular conjunctival repair material was 99.87%.

[0081] Table 1 Detection results of α-Gal antigen content in samples

[0082]

[0083] *: (Number of α-Gal antigen epitopes in porcine eye conjunctiva - Number of α-Gal antigen epitopes in acellular conjunctival repair material) / Number of α-Gal antigen epitopes in porcine eye conjunctiva × 100.

[0084] (3) DNA residual detection: According to the method of YY / T 0606.25-2014 "Tissue Engineering Medical Products - Part 25: Determination of DNA Residual Amount in Animal-derived Biomaterials - Fluorescent Staining Method", the DNA residual amounts of porcine conjunctival tissue and the acellular conjunctival repair material obtained in Example 1 were determined. The main experimental procedures included: sample collection and enzymatic digestion, DNA purification. A double-stranded DNA fluorescent dye was used to specifically bind to double-stranded DNA to form a complex. A fluorescence microplate reader was used to detect the ultra-strong fluorescence signal generated under excitation at a wavelength of 480 nm, with an emission wavelength of 520 nm and a cut-off wavelength of 530 nm. Within a certain DNA concentration range and when the fluorescent dye was in excess, the fluorescence intensity was proportional to the DNA concentration. According to the fluorescence intensity of the test sample, the DNA residual amount in the test sample was calculated, and 3 parallel samples were set up.

[0085] DNA residual detection results: Sample 1 was 0.87 ng / mg, Sample 2 was 0.96 ng / mg, and Sample 3 was 0.81 ng / mg. The average was 0.88 ng / mg, which was much lower than the standard requirement that the DNA residual amount of products made from common animal-derived materials should be less than 50 ng / mg.

[0086] (4) Tensile force and suture tearing force detection

[0087] 1) Tensile strength detection: Porcine conjunctival tissue, uncrosslinked acellular conjunctival repair material, and the acellular conjunctival repair material obtained in Example 1 were taken, soaked in physiological saline for 1 minute, taken out, and cut into rectangles of 3 mm × 7 mm. An electronic tensile testing machine was used to measure the tensile strength of the samples. The two sides of the samples were clamped and fixed on the clamps (the longitudinal axis of the specimen coincided with the center line connecting the upper and lower clamps, with appropriate tightness). They were stretched at a stable speed of 50 mm / min until fracture, and the maximum load force at the time of sample fracture was recorded. At the same time, porcine conjunctival tissue (without any treatment) and non-acellular porcine conjunctival tissue material (the preparation method is described in Example 1 of Patent CN105770994A) were set up. The maximum load forces of the three groups of samples, namely porcine conjunctival tissue, non-acellular porcine conjunctival tissue material, and the acellular conjunctival repair material prepared by the present invention, were 2.2 N, 2.1 N, and 3.5 N respectively.

[0088] The detection results showed that the tensile strength of the samples prepared by this method was significantly improved (see attachment Figure 2 ).

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

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

[0091] Example 5

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

[0093] (1) HE staining experiment: The acellular conjunctival repair material obtained in Example 2 and the acellular porcine conjunctival tissue are stained by HE staining according to the conventional method. The results are shown in Figure 4 As shown, the xenogeneic cells in the acellular conjunctival repair material are completely removed, the extracellular collagen fiber matrix structure is retained, and the original tissue structure of the conjunctiva remains intact.

[0094] (2) DNA residual detection: According to the method of YY / T 0606.25-2014 "Tissue Engineering Medical Products - Part 25: Determination of DNA Residual Amount in Animal-derived Biomaterials - Fluorescent Staining Method", the DNA residual amounts of porcine conjunctival tissue and the acellular conjunctival repair material obtained in Example 1 were determined. The main experimental procedures included: sample preparation and enzymatic digestion, DNA purification. A double-stranded DNA fluorescent dye was used to specifically bind to double-stranded DNA to form a complex. A fluorescence microplate reader was used to detect the ultra-strong fluorescence signal generated under excitation at a wavelength of 480 nm, with an emission wavelength of 520 nm and a cut-off wavelength of 530 nm. Within a certain DNA concentration range and when the fluorescent dye was in excess, the fluorescence intensity was proportional to the DNA concentration. According to the fluorescence intensity of the test sample, the DNA residual amount in the test sample was calculated. DNA residual detection results: Sample 1 was 0.57 ng / mg, Sample 2 was 0.55 ng / mg, and Sample 3 was 0.53 ng / mg. The average was 0.55 ng / mg, which was much less than the standard requirement that the DNA residual amount of products made from common animal-derived materials should be less than 50 ng / mg.

[0095] (3) Tensile force and suture tearing force detection

[0096] 1) Tensile strength detection: Take porcine conjunctival tissue and the acellular conjunctival repair material obtained in Example 2, soak it in physiological saline for 1 minute, take it out, and cut it into rectangles of 3 mm × 7 mm. Use an electronic tensile machine to measure the tensile strength of the sample. The two sides of the sample were clamped and fixed on the fixture with clamps (the longitudinal axis of the specimen coincided with the center line connecting the upper and lower clamps, with appropriate tightness). Stretch at a stable speed of 50 mm / min until it breaks, and record the maximum load force when the sample breaks. At the same time, set up porcine conjunctival tissue (untreated) and non-acellular porcine conjunctival tissue material (the preparation method is described in Example 1 of Patent CN105770994A). The maximum load forces of the three groups of samples, namely porcine conjunctival tissue, non-acellular porcine conjunctival tissue material, and the acellular conjunctival repair material prepared by the present invention, were 2.5 N, 2.5 N, and 3.6 N respectively. The detection results showed that the tensile strength of the samples prepared by this method was significantly improved (see attached Figure 5 ).

[0097] 2) Suture tearing force detection: Take porcine conjunctival tissue and the acellular conjunctival repair material obtained in Example 2, soak it in physiological saline for 1 minute, take it out, cut it into a square of 5 mm × 5 mm, and pass a 10-0 nylon suture through the center point of the product. Use an electronic tensile machine to measure the suture tearing force of the sample. Fix one end of the sample to the fixture, and fix the tail end of the suture after it freely hangs down to ensure that the experimental sample is not stretched or damaged by the fixture. The suture is stretched at a stable speed of 50 mm / min, and record the magnitude of the pulling force that pulls the suture out of the sample or damages the sample. At the same time, set up porcine conjunctival tissue (untreated) and acellular porcine conjunctival tissue material (the preparation method is described in Example 1 of Patent CN105770994A). The pulling forces of the three groups of samples, namely porcine conjunctival tissue, acellular porcine conjunctival tissue material, and the acellular conjunctival repair material prepared by the present invention, are 0.73 N, 0.78 N, and 1.22 N respectively. The detection results show that the suture tearing force of the samples prepared by this method has been significantly improved (see attached Figure 6 ).

[0098] Example 6

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

[0100] Free the partial bulbar conjunctiva on the left side of the right rectus muscle of a healthy New Zealand white rabbit on the ocular surface (with an area of about 10 mm × 6 mm) to construct a conjunctival defect model. Randomly divide it into a test group and a control group. The test group transplants the acellular conjunctival tissue obtained in Example 1 at the defect site, and the control group transplants biological amniotic membrane at the defect site.

[0101] Two weeks after transplantation, take pictures of the partial bulbar conjunctiva on the left side of the right rectus muscle of the two groups of rabbits, and stain the sections of the repaired conjunctival tissue by the HE method to observe the sections.

[0102] The results show that: two weeks after transplantation, the acellular conjunctival repair material in the test group is in place, and the boundary between the conjunctival repair material and the normal conjunctiva basically disappears, and the wound surface is repaired (see attached Figure 7 , attached Figure 8 ); two weeks after transplantation, the conjunctival repair material in the control group begins to dissolve, and it is observed that the conjunctival tissue is severely congested in the figure, and the number of purple-red inflammatory cells increases and aggregates in the HE staining, indicating that a certain inflammatory reaction has occurred (see attached Figure 9 , attached Figure 10 ).

[0103] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A preparation method of a conjunctival repair material, characterized in that, Comprising the following steps: Performing ultrasonic treatment on the separated animal conjunctival tissue to obtain ultrasonically treated conjunctival tissue; Performing decellularization treatment on the ultrasonically treated conjunctival tissue under the action of nuclease to obtain decellularized conjunctival tissue; Performing ultra-high hydrostatic pressure treatment crosslinking on the decellularized conjunctival tissue in the presence of a crosslinking agent to obtain a conjunctival repair material; the crosslinking agent includes any one of the following substances: carbodiimide, genipin, epigallocatechin gallate, and proanthocyanidin; During the crosslinking, the pressure of the ultra-high hydrostatic pressure treatment is 400 - 800 MPa, and the time of the ultra-high hydrostatic pressure treatment is 5 - 10 min.

2. The preparation method according to claim 1, characterized in that, The mass concentration of the crosslinking agent is 0.1% - 5%.

3. The preparation method according to claim 1, wherein The nuclease includes at least one of DNase, RNase, and recombinant nuclease; The final concentration of the nuclease is 50 - 1500 U / ml.

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

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

6. According to the preparation method described in claim 1, wherein, The frequency of the ultrasonic treatment is 20 - 60 Hz, and the time of the ultrasonic treatment is 2 - 20 min.

7. The preparation method according to any one of claims 1 to 6, characterized in that, The animal includes any one of the following: pig, cow, and sheep.

8. The conjunctival repair material prepared by the preparation method according to any one of claims 1 to 7, characterized in that, The suture tearing force is not less than 1.0 N; the tensile strength is not less than 3.0 N.

9. Use of the conjunctival repair material prepared by the preparation method according to claim 8 in the preparation of an engineering material for conjunctival injury repair.

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