Dry amnion as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medical materials, and particularly relates to a dry amnion as well as a preparation method and application thereof. The method comprises the following steps: carrying out atomization spraying treatment on a pretreated amnion sample by adopting a hydrophilic hydrogen bond supplement reagent, and then carrying out steam crosslinking by adopting a crosslinking agent; finally, the hydrophilic hydrogen bond supplementary reagent is adopted for atomization spraying treatment again, and the dry amnion is obtained. According to the non-freeze-drying steam crosslinking dry amnion preparation method provided by the invention, hydrophilic hydrogen bond supplementary reagent treatment is carried out before and after the atomization spraying crosslinking step, and an atomization spraying non-soaking treatment mode is adopted; the beneficial components, including water-soluble growth factors and cell factors, of the amniotic membrane are preserved in a mode of treating the amniotic membrane through steam crosslinking, and the problem that the amniotic membrane is only suitable for small-area tissue repair surgery at present and cannot be popularized and used due to the fact that the degradation rate is too high and tearing and damage are prone to occurring in clinical application is solved.

Description

Technical Field

[0001] This application belongs to the technical field of medical materials, and specifically relates to a dry amniotic membrane and its preparation method and application. Background Art

[0002] The amniotic membrane is a thin and transparent tissue mainly composed of fibrin, collagen, glycosaminoglycans, etc., and has good biocompatibility and low immunogenicity. The amniotic membrane has various biological functions such as protection, covering, repair, and anti-infection, and has been widely used in clinical applications such as ophthalmic surgery (such as corneal repair, corneal transplantation), burn treatment, covering and healing of chronic wounds, and placenta transplantation. It is used as a natural "substitute material" in medicine, providing an ideal environment for tissue repair.

[0003] Although the amniotic membrane has significant advantages in clinical applications, its mechanical properties still have certain limitations. The strength and toughness of the amniotic membrane are relatively low, and it is prone to rupture or failure under external forces such as stretching and shearing. This makes the amniotic membrane likely to be unstable under greater mechanical stress or during long-term use, limiting its use in high-strength applications. In order to further improve the application effect of the amniotic membrane, enhancing its mechanical properties has become a key research direction. The existing methods for storing amniotic membrane products are mainly ultra-low temperature (-75°C to 85°C) or freeze-drying storage. Ultra-low temperature storage of amniotic membrane requires cold chain transportation and the equipped ultra-low temperature refrigerator, and freeze-dried amniotic membrane is brittle, resulting in limited promotion and application.

[0004] In the existing amniotic membrane cross-linking technologies, the amniotic membrane is soaked in the prepared cross-linking agent solution, which inevitably leads to the loss of the beneficial components of the amniotic membrane itself, including water-soluble growth factors and cytokines, reducing its biological activity.

[0005] In summary, it is urgent to develop a new preparation method to improve the mechanical strength of the amniotic membrane and reduce the loss of beneficial components such as growth factors and cytokines. Summary of the Invention

[0006] The purpose of the present invention is to solve the deficiencies of the prior art, and provide a dry amniotic membrane and its preparation method and application, specifically adopting the following technical solutions: In the first aspect, the present invention provides a method for preparing a dry amniotic membrane based on steam cross-linking, including the following steps: S1. Atomizing and spraying the pretreated amniotic membrane sample with a hydrophilic hydrogen bond supplement reagent, and standing still; S2. Steam cross-linking the standing amniotic membrane sample with a cross-linking agent; S3. Atomizing and spraying the steam-cross-linked amniotic membrane sample with the hydrophilic hydrogen bond supplement reagent again, and standing still to obtain the dry amniotic membrane; The hydrophilic hydrogen bond replenishing reagent includes one or more of compounds containing oxygen-containing groups and compounds containing nitrogen-containing groups; The crosslinking agent includes glutaraldehyde solution.

[0007] In the above preparation method, the treatment with the hydrophilic hydrogen bond replenishing reagent is carried out before and after the atomized spraying crosslinking step, and the non-immersion treatment method of atomized spraying is adopted. The significance of this treatment method compared with the traditional immersion treatment method is that it can prevent the weakening of hydrogen bond action caused by natural air drying after the amniotic membrane shed is fixed, and then irreversible structural collapse occurs and the mechanical strength decreases. According to the experimental results of the present invention: (1) If the atomized spraying treatment of the hydrophilic hydrogen bond replenishing reagent is not carried out before and after the amniotic membrane crosslinking, the amniotic membrane will have its hydrogen bond action weakened due to natural air drying, and then the amniotic membrane structure will undergo irreversible collapse and its mechanical strength will decrease. That is, from the uniaxial tensile test results of the amniotic membrane, the mechanical strength of the amniotic membrane is significantly improved, from 1.19 MPa (Comparative Example 7) to 4.21 MPa (Example 3), and the improvement amplitude reaches 253.78%.

[0008] (2) If the atomized spraying treatment of the hydrophilic hydrogen bond replenishing reagent is carried out only before or after the amniotic membrane crosslinking, it will also cause a decrease in its mechanical strength. That is, from the uniaxial tensile test results of the amniotic membrane, compared with the amniotic membranes treated with the hydrogen bond replenishing reagent only before or after crosslinking, the mechanical strengths of the amniotic membranes prepared in Comparative Example 8 and Comparative Example 9 are only improved by 27.73% and 1.68% respectively compared with the naturally air-dried amniotic membrane in Comparative Example 7. However, for the amniotic membrane of the preferred embodiment prepared by the method of the present invention, the mechanical strength of Example 3 in the present invention is improved by 253.78% compared with Comparative Example 7.

[0009] As a further preferred implementation manner, the pretreatment includes: Wash the surface impurities of the fresh amniotic membrane with water and then fix it.

[0010] As a further preferred implementation manner, the specific steps of the steam crosslinking are as follows: Place the crosslinking agent in a dryer, and place the amniotic membrane sample 5 cm - 15 cm above the crosslinking agent, and crosslink for 24 h - 48 h. Turn the amniotic membrane sample over every 12 h or 24 h.

[0011] As a further preferred implementation manner, the hydrophilic hydrogen bond replenishing reagent includes one or more of glycerol, ethanol, tyrosine solution, carboxymethyl chitosan solution, and hyaluronic acid solution.

[0012] As a further preferred implementation manner, the hydrophilic hydrogen bond replenishing reagent is a mixed solution of glycerol and ethanol.

[0013] As a further preferred embodiment, the volume ratio of the glycerol to the ethanol is 2:8.

[0014] Among them, glycerol (C 3 H 8 O 3 ): Each molecule contains 3 hydroxyl groups, can form multiple hydrogen bonds with water, and has strong hydrophilicity; ethanol (C 2 H 5 OH): Each molecule contains 1 hydroxyl group, can also form hydrogen bonds with water, but its hydrophilicity is weaker than that of glycerol. Since the viscosity of glycerol is relatively high, in the present invention, it is necessary to prepare a mixed solution with a low viscosity capable of atomizing amniotic membrane in a certain ratio with ethanol. On the premise of meeting the requirement of being able to atomize the amniotic membrane, the prepared solution is controlled by adjusting the ratio, thereby further optimizing its hydrophilicity and other physical and chemical properties. The preferred ratio of the glycerol-ethanol mixed solution in the present invention is 2:8.

[0015] As a further preferred embodiment, the concentration of the tyrosine solution is 0.5% - 2%, the concentration of the carboxymethyl chitosan solution is 0.5% - 5%, and the concentration of the hyaluronic acid solution is 0.5% - 2%.

[0016] Among them, carboxymethyl chitosan (CMCS) is a chemically modified chitosan, which has good hydrophilicity and biocompatibility. Since a large number of carboxyl groups (-COOH) are contained in its molecular chain, CMCS can form hydrogen bonds with water molecules, thereby enhancing its hydrophilicity; hyaluronic acid (HA) is a high-molecular polysaccharide, which is widely present in human connective tissues, skin and eyeballs. Due to the presence of a large number of hydroxyl groups and carboxyl groups in its molecular chain, hyaluronic acid has excellent hydrophilicity and moisturizing properties, and can form hydrogen bonds with water molecules, thereby enhancing its hydrophilicity. Tyrosine (Tyr) is an important amino acid, and its side chain contains a phenolic hydroxyl group (-OH) and a benzene ring. Due to the presence of the phenolic hydroxyl group, tyrosine has certain hydrophilicity and can form hydrogen bonds with water molecules, so it can be used as a hydrophilic hydrogen bond supplement reagent.

[0017] As a further preferred embodiment, the concentration of the glutaraldehyde solution is 5% - 25%.

[0018] In the second aspect, the present invention provides a dry amniotic membrane prepared by the above method.

[0019] In the third aspect, the present invention provides the application of the above dry amniotic membrane in the preparation of materials for promoting tissue repair and regeneration.

[0020] Compared with the prior art, the present invention has at least the following beneficial effects: (1) By means of steam cross - linking treatment of amniotic membrane, the beneficial components of the amniotic membrane itself, including water - soluble growth factors and cytokines, are preserved, avoiding the loss of the beneficial components of the amniotic membrane caused by cross - linking the amniotic membrane by soaking in the existing patented technology, which is beneficial to maintaining the biological activity of the amniotic membrane itself.

[0021] (2) The treatment with hydrophilic hydrogen - bond supplement reagent in the present invention is carried out before and after the atomized spraying cross - linking step, and the non - soaking treatment method of atomized spraying is adopted. The special feature and significance of this treatment method are as follows: it can prevent the weakening of hydrogen - bond action caused by natural air - drying after the amniotic membrane plank is fixed, and then irreversible structural collapse and a decrease in mechanical strength occur.

[0022] (3) After the steam cross - linking treatment in the present invention, the mechanical strength of the amniotic membrane is improved: from the results of the uniaxial tensile mechanical test of the amniotic membrane, for the amniotic membrane of the preferred embodiment prepared by the method described in the present invention, compared with the fresh one, the maximum tensile fracture stress is increased from 0.56 MPa to 2.21 MPa, that is, it is increased by 294.64%. To a certain extent, it solves the problem that it is easy to tear and break during clinical application, resulting in its current only being applicable to small - area tissue repair surgeries.

[0023] (4) After the steam cross - linking treatment in the present invention, the degradation degree of the amniotic membrane is reduced: from the results of the collagenase degradation test of the amniotic membrane, for the amniotic membrane of the preferred embodiment prepared by the method described in the present invention, compared with the fresh one, the weight loss rate of enzyme degradation is reduced from 100% to 77.97%. To a certain extent, it solves the problem that the degradation rate of the amniotic membrane in the body is too fast during clinical application, resulting in its inability to be widely used. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the following - described drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0025] Figure 1 Shown is the flow schematic diagram of the preparation method in the present invention; Figure 2 Shown is the thickness - measuring schematic diagram of the thickness - gauge in the uniaxial tensile mechanical test in the present invention; Figure 3 Shown is the uniaxial tensile mechanical test schematic diagram in the present invention; Figure 4 Shown are the schematic diagrams of each amniotic membrane (Examples 1 - 3 and Comparative Examples 2 - 4) before the collagenase degradation test in the present invention; Figure 5The following shows the degradation schematic diagrams of each amniotic membrane of Examples 1-3 and Comparative Examples 2-4 of the present invention after the collagenase degradation test; Figure 6 The following shows the comparison chart of the uniaxial tensile mechanical test of the amniotic membrane in Examples 1-6 and Tbilisi 1-9; Figure 7 The following shows the comparison chart of the collagenase degradation test of the amniotic membrane in Examples 1-6 and Tbilisi 1-9. Detailed implementation manners

[0026] Next, the technical solutions in the embodiments of the present application will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present application.

[0027] Example 1 A preparation method of non-freeze-dried steam-crosslinked dry amniotic membrane (the flow chart is as shown), including the following steps: Figure 1 (1) Cleaning: Take fresh amniotic membrane, put it into a 500 mL beaker, and wash it 5 times with sterile water; (2) Fixing: Cut the washed amniotic membrane into 10×10 cm, flatten it and place it in a 10×8 cm stretching board, clamp and fix it, and tie it tightly around with a yellow rubber band; (3) Preparing a hydrophilic hydrogen bond replenishing reagent solution: Take 20 mL of glycerol and 80 mL of ethanol and place them in a 500 mL beaker. Place a stir bar in the beaker, stir for 10 min, and then put the prepared solution into a spray bottle; (4) Dry state treatment: For the stretching board fixed with amniotic membrane, hold the spray bottle filled with the prepared hydrophilic hydrogen bond replenishing solution 25 cm away from the stretching board fixed with amniotic membrane, press the nozzle, and continuously spray the front and back sides of the amniotic membrane three times respectively, and then place it on an 8.5 cm triangular iron stand and leave it at room temperature for 8 hours to complete the atomization spraying process of the hydrophilic hydrogen bond replenishing reagent, that is, the dry state treatment; (5) Amniotic membrane crosslinking treatment: Prepare a 5% glutaraldehyde solution, that is, 2 mL of glutaraldehyde (50% w / v) and 20 mL of sterile water; Put the prepared solution at the bottom of the dryer, put the amniotic membrane after dry state treatment on the upper part of the dryer, close it and put it into an incubator, react at 45°C for 24 hours, and turn it over every 12 hours; That is, the crosslinking treatment of the amniotic membrane is completed; (6) Dry state treatment after amniotic membrane crosslinking: This step is the same as step (4) to obtain a non-freeze-dried steam-crosslinked dry amniotic membrane. (6) Dry state treatment after amniotic membrane crosslinking: This step is the same as step (4), and a non-freeze-dried steam-crosslinked dry amniotic membrane is obtained.

[0028] Example 2 A preparation method of non-freeze-dried steam-crosslinked dry amniotic membrane. The specific process of this example is similar to that of Example 1, except that step (5) in Example 1 is as follows. The specific step (5) in this example is: Crosslinking treatment of amniotic membrane: Prepare a 15% glutaraldehyde solution, that is, 4 mL of glutaraldehyde (50% w / v) and 20 mL of sterile water. Place the prepared solution at the bottom of the dryer, place the dry-treated amniotic membrane at the upper part of the dryer, seal it and put it into the incubator, and react at 45 °C for 24 hours, turning it over every 12 hours, that is, the crosslinking treatment of the amniotic membrane is completed; other steps are the same as those in Example 1.

[0029] Example 3 A preparation method of non-freeze-dried steam-crosslinked dry amniotic membrane. The specific process of this example is similar to that of Example 1, except that step (5) in Example 1 is as follows. The specific step (5) in this example is: Crosslinking treatment of amniotic membrane: Prepare a 25% glutaraldehyde solution, that is, 10 mL of glutaraldehyde (50% w / v) and 20 mL of sterile water. Place the prepared solution at the bottom of the dryer, place the dry-treated amniotic membrane at the upper part of the dryer, seal it and put it into the incubator, and react at 45 °C for 24 hours, turning it over every 12 hours, that is, the crosslinking treatment of the amniotic membrane is completed; other steps are the same as those in Example 1.

[0030] Example 4 A preparation method of non-freeze-dried steam-crosslinked dry amniotic membrane. The specific process of this example is similar to that of Example 1, except that step (3) in Example 1 is as follows. The specific step (3) in this example is: Prepare hydrophilic hydrogen bond supplement reagent solution: Prepare 50 mL of 1% (w / v) tyrosine solution, and put the prepared solution into a spray bottle; other steps are the same as those in Example 1.

[0031] Example 5 A preparation method of non-freeze-dried steam-crosslinked dry amniotic membrane. The specific process of this example is similar to that of Example 1, except that step (3) in Example 1 is as follows. The specific step (3) in this example is: Prepare hydrophilic hydrogen bond supplement reagent solution: Prepare 50 mL of 2.5% (w / v) carboxymethyl chitosan solution, and put the prepared solution into a spray bottle; other steps are the same as those in Example 1.

[0032] Example 6 A preparation method of non-freeze-dried steam-crosslinked dry amniotic membrane. The specific process of this example is similar to that of Example 1, except that step (3) in Example 1 is as follows. The specific step (3) in this example is: Preparation of hydrophilic hydrogen bond supplementary reagent solution: Prepare 50 mL of 1% (w / v) hyaluronic acid solution and put the prepared solution into a spray bottle; other steps are the same as those in Example 1.

[0033] Comparative Example 1 In this Example 1, fresh amniotic membrane without any treatment was used. Only the washed amniotic membrane was tightened with a stretching board, and the four sides of the stretching board were tied with yellow rubber bands to fix the fresh amniotic membrane to keep it flat for standby as a comparison.

[0034] Comparative Example 2 In this example, naturally air-dried amniotic membrane without cross-linking and atomized spraying treatment with hydrophilic hydrogen bond supplementary reagent was used. Only the washed amniotic membrane was tightened with a stretching board, and the four sides of the stretching board were tied with yellow rubber bands to fix the fresh amniotic membrane to keep it flat, and it was placed on a triangular iron stand and air-dried at room temperature as a comparison.

[0035] Comparative Example 3 A preparation method of non-freeze-dried dry amniotic membrane (without cross-linking), the specific process is as follows: (1) Cleaning: Take fresh amniotic membrane, put it into a 500 mL beaker, and wash it 5 times with sterile water; (2) Fixing: Cut the washed amniotic membrane into 10×10 cm, flatten it and place it in a 10×8 cm stretching board, clamp and fix it and tie the four sides tightly with yellow rubber bands; (3) Preparation of hydrophilic hydrogen bond supplementary reagent solution: Prepare 50 mL of a mixed solution of glycerol and ethanol with a volume ratio of 2:8, and put the prepared solution into a spray bottle; (4) Dry state treatment: For the stretching board fixed with amniotic membrane, place the spray bottle filled with the prepared hydrophilic hydrogen bond supplementary solution at a distance of 25 cm from the stretching board fixed with amniotic membrane, press the nozzle, spray the front and back sides of the amniotic membrane three times continuously, and then place it on an 8.5 cm triangular iron stand and let it stand overnight at room temperature, that is, complete the atomized spraying process of the hydrophilic hydrogen bond supplementary reagent, that is, the dry state treatment, to obtain dry amniotic membrane.

[0036] Comparative Example 4 A preparation method of non-freeze-dried steam cross-linked dry amniotic membrane, the specific process is as follows: (1) Cleaning: Take fresh amniotic membrane, put it into a 500 mL beaker, and wash it 5 times with sterile water; (2) Fixing: Cut the washed amniotic membrane into 10×10 cm, flatten it and place it in a 10×8 cm stretching board, clamp and fix it and tie the four sides tightly with yellow rubber bands; (3) Preparation of hydrophilic hydrogen bond supplementary reagent solution: Prepare 50 mL of 1% (w / v) hyaluronic acid solution, and put the prepared solution into a spray bottle; (4)Dry state treatment: For the board fixed with amniotic membrane, hold the spray bottle filled with the prepared hydrophilic hydrogen bond replenishing solution at a distance of 25 cm from the board fixed with amniotic membrane, press the nozzle, and continuously spray the front and back sides of the amniotic membrane three times respectively. Then place it on an 8.5 cm triangular iron stand and leave it at room temperature overnight, thus completing the atomizing spraying process of the hydrophilic hydrogen bond replenishing reagent, that is, the dry state treatment; (5)Amniotic membrane crosslinking treatment: Prepare a 1% glutaraldehyde solution, that is, 0.4 ml of glutaraldehyde (50% w / v) and 20 ml of sterile water. Put the prepared solution at the bottom of the desiccator, put the amniotic membrane after dry state treatment at the upper part of the desiccator, seal it and put it in the incubator, and react at 45 °C for 24 hours, and turn it over once every 12 hours. That is, the crosslinking treatment of the amniotic membrane is completed; (6)Dry state treatment after amniotic membrane crosslinking: This step is the same as step (4), and the dry amniotic membrane with non-freeze-dried steam crosslinking is obtained.

[0037] Comparative Example 5 A preparation method of non-freeze-dried steam crosslinked dry amniotic membrane (crosslinking agent immersion treatment), the specific process is as follows: (1)Cleaning: Take fresh amniotic membrane and put it into a 500 ml beaker, and wash it 5 times with sterile water; (2)Fixing: Cut the washed amniotic membrane into 10×10 cm, flatten it and place it in a 10×8 cm board, clamp and fix it and tie the four sides tightly with a yellow rubber band; (3)Prepare hydrophilic hydrogen bond replenishing reagent solution: Prepare 50 mL of 1% (w / v) hyaluronic acid solution, and put the prepared solution into a spray bottle; (4)Dry state treatment: For the board fixed with amniotic membrane, hold the spray bottle filled with the prepared hydrophilic hydrogen bond replenishing solution at a distance of 25 cm from the board fixed with amniotic membrane, press the nozzle, and continuously spray the front and back sides of the amniotic membrane three times respectively. Then place it on an 8.5 cm triangular iron stand and leave it at room temperature overnight, thus completing the atomizing spraying process of the hydrophilic hydrogen bond replenishing reagent, that is, the dry state treatment; (5)Amniotic membrane crosslinking treatment: Prepare a 1% glutaraldehyde solution, that is, 0.4 ml of glutaraldehyde (50% w / v) and 20 ml of sterile water. Put the amniotic membrane after dry state treatment into the prepared solution and react at room temperature for 1 hour, then take it out and place it until the surface is dry; (6)Dry state treatment after amniotic membrane crosslinking: This step is the same as step (4), and the dry amniotic membrane with non-freeze-dried steam crosslinking is obtained.

[0038] Comparative Example 6 A preparation method of freeze-dried steam crosslinked dry amniotic membrane (dry amniotic membrane obtained by freeze-drying without treatment with hydrophilic hydrogen bond replenishing reagent), the specific process is as follows: (1) Cleaning: Take fresh amniotic membrane, put it into a 500 mL beaker, and wash it 5 times with sterile water; (2) Fixing: Cut the washed amniotic membrane into 10×10 cm, flatten it and place it in a 10×8 cm stretching board, clamp and fix it, and tie the four sides tightly with a yellow rubber band; (3) Amniotic membrane crosslinking treatment: Prepare a 5% glutaraldehyde solution, that is, 2 mL of glutaraldehyde (50% w / v) and 20 mL of sterile water; Put the prepared solution at the bottom of the dryer, put the dry-treated amniotic membrane at the upper part of the dryer, seal it and put it into the incubator, react at 45 °C for 24 hours, and turn it over every 12 hours; That is, the crosslinking treatment of the amniotic membrane is completed.

[0039] (4) Freeze-drying of amniotic membrane: Freeze-dry the amniotic membrane after steam crosslinking is completed, control the freezing temperature at 40 °C, and the drying time is 24 - 96 h to obtain freeze-dried steam crosslinked dry amniotic membrane.

[0040] Comparative Example 7 A preparation method of steam crosslinked dry amniotic membrane (dry amniotic membrane obtained by natural air drying without treatment with hydrophilic hydrogen bond supplement reagent), the specific process is as follows: (1) Cleaning: Take fresh amniotic membrane, put it into a 500 mL beaker, and wash it 5 times with sterile water; (2) Fixing: Cut the washed amniotic membrane into 10×10 cm, flatten it and place it in a 10×8 cm stretching board, clamp and fix it, and tie the four sides tightly with a yellow rubber band; (3) Amniotic membrane crosslinking treatment: Prepare a 5% glutaraldehyde solution, that is, 2 mL of glutaraldehyde (50% w / v) and 20 mL of sterile water; Put the prepared solution at the bottom of the dryer, put the dry-treated amniotic membrane at the upper part of the dryer, seal it and put it into the incubator, react at 45 °C for 24 hours, and turn it over every 12 hours; That is, the crosslinking treatment of the amniotic membrane is completed (4) Natural air drying of amniotic membrane: Place the steam crosslinked amniotic membrane at room temperature and wait for it to air dry naturally to obtain steam crosslinked dry amniotic membrane.

[0041] Comparative Example 8 A preparation method of non-freeze-dried steam crosslinked dry amniotic membrane (only atomizing spray of hydrophilic hydrogen bond supplement reagent before crosslinking), including the following steps: (1) Cleaning: Take fresh amniotic membrane, put it into a 500 mL beaker, and wash it 5 times with sterile water; (2) Fixing: Cut the washed amniotic membrane into 10×10 cm, flatten it and place it in a 10×8 cm stretching board, clamp and fix it, and tie the four sides tightly with a yellow rubber band; (3) Preparation of hydrophilic hydrogen bond replenishing reagent solution: Take 20 mL of glycerol and 80 mL of ethanol and place them in a 500 mL beaker. Place a magnetic stir bar in the beaker and stir for 10 min. Then, put the prepared solution into a spray bottle. (4) Dry state treatment: For the stretching board fixed with amniotic membrane, hold the spray bottle filled with the prepared hydrophilic hydrogen bond replenishing solution at a distance of 25 cm from the stretching board fixed with amniotic membrane, press the nozzle, and continuously spray the front and back sides of the amniotic membrane three times respectively. Then, place it on an 8.5 cm triangular iron stand and leave it at room temperature overnight, thus completing the atomizing spraying process of the hydrophilic hydrogen bond replenishing reagent, which is also the dry state treatment. (5) Amniotic membrane crosslinking treatment: Prepare a 5% glutaraldehyde solution, that is, 2 mL of glutaraldehyde (50% w / v) and 20 mL of sterile water. Put the prepared solution at the bottom of a desiccator, put the amniotic membrane after dry state treatment at the upper part of the desiccator, seal it and put it into an incubator, and react at 45 °C for 24 hours, with turning over once every 12 hours. Thus, the crosslinking treatment of the amniotic membrane is completed, and a dry amniotic membrane with non-freeze-dried steam crosslinking is obtained.

[0042] Comparative Example 9 A preparation method of a non-freeze-dried steam crosslinked dry amniotic membrane (only atomizing spraying of the hydrophilic hydrogen bond replenishing reagent is carried out after crosslinking), including the following steps: (1) Cleaning: Take fresh amniotic membrane and put it into a 500 mL beaker, and wash it 5 times with sterile water. (2) Fixing: Cut the washed amniotic membrane into 10×10 cm, flatten it and place it in a 10×8 cm stretching board, clamp and fix it, and tie the four sides tightly with a yellow rubber band. (3) Amniotic membrane crosslinking treatment: Prepare a 5% glutaraldehyde solution, that is, 2 mL of glutaraldehyde (50% w / v) and 20 mL of sterile water. Put the prepared solution at the bottom of a desiccator, put the amniotic membrane after dry state treatment at the upper part of the desiccator, seal it and put it into an incubator, and react at 45 °C for 24 hours, with turning over once every 12 hours. Thus, the crosslinking treatment of the amniotic membrane is completed. (4) Preparation of hydrophilic hydrogen bond replenishing reagent solution: Prepare a hydrophilic hydrogen bond replenishing reagent solution: Take 20 ml of glycerol and 80 ml of ethanol and place them in a 500 ml beaker. Place a magnetic stir bar in the beaker and stir for 10 min. Then, put the prepared solution into a spray bottle.

[0043] (5) For the stretching board fixed with amniotic membrane, hold the spray bottle filled with the prepared hydrophilic hydrogen bond replenishing solution at a distance of 25 cm from the stretching board fixed with amniotic membrane, press the nozzle, and continuously spray the front and back sides of the amniotic membrane three times respectively. Then, place it on an 8.5 cm triangular iron stand and leave it at room temperature overnight, thus obtaining a non-freeze-dried steam crosslinked dry amniotic membrane after completing the atomizing spraying process of the hydrophilic hydrogen bond replenishing reagent.

[0044] Comparative Example 10 A preparation method of non-freeze-dried steam-crosslinked dry amniotic membrane (high-concentration hydrogen bond supplement reagent), comprising the following steps: (1) Cleaning: Take fresh amniotic membrane, put it into a 500 mL beaker, and wash it 5 times with sterile water; (2) Fixing: Cut the cleaned amniotic membrane into 10×10 cm, flatten it and place it in a 10×8 cm stretching board, clamp and fix it, and tie the four sides tightly with a yellow rubber band; (3) Preparing a hydrophilic hydrogen bond supplement reagent solution: Prepare 50 mL of 2.5% (w / v) hyaluronic acid solution, and put the prepared solution into a spray bottle; (4) Dry state treatment: For the stretching board with the amniotic membrane fixed, place the spray bottle filled with the prepared hydrophilic hydrogen bond supplement solution 25 cm away from the stretching board with the amniotic membrane fixed, press the nozzle, spray the front and back sides of the amniotic membrane three times continuously, and then place it on an 8.5 cm triangular iron stand and leave it at room temperature for 8 hours to complete the atomization spraying process of the hydrophilic hydrogen bond supplement reagent, that is, the dry state treatment; (5) Amniotic membrane crosslinking treatment: Prepare a 5% glutaraldehyde solution, that is, 2 mL of glutaraldehyde (50% w / v) and 20 mL of sterile water; put the prepared solution at the bottom of the dryer, put the amniotic membrane after dry state treatment in the upper part of the dryer, seal it and put it in an incubator, react at 45 °C for 24 hours, and turn it over every 12 hours; that is, complete the crosslinking treatment of the amniotic membrane; (6) Dry state treatment after amniotic membrane crosslinking: This step is the same as step (4) to obtain a non-freeze-dried steam-crosslinked dry amniotic membrane.

[0045] Comparative Example 11 A preparation method of non-freeze-dried steam-crosslinked dry amniotic membrane (ultra-low-concentration hydrogen bond supplement reagent), comprising the following steps: (1) Cleaning: Take fresh amniotic membrane, put it into a 500 mL beaker, and wash it 5 times with sterile water; (2) Fixing: Cut the cleaned amniotic membrane into 10×10 cm, flatten it and place it in a 10×8 cm stretching board, clamp and fix it, and tie the four sides tightly with a yellow rubber band; (3) Preparing a hydrophilic hydrogen bond supplement reagent solution: Prepare 50 mL of 0.1% (w / v) hyaluronic acid solution, and put the prepared solution into a spray bottle; (4) Dry state treatment: For the stretching board with the amniotic membrane fixed, place the spray bottle filled with the prepared hydrophilic hydrogen bond supplement solution 25 cm away from the stretching board with the amniotic membrane fixed, press the nozzle, spray the front and back sides of the amniotic membrane three times continuously, and then place it on an 8.5 cm triangular iron stand and leave it at room temperature for 8 hours to complete the atomization spraying process of the hydrophilic hydrogen bond supplement reagent, that is, the dry state treatment; (5)Amniotic membrane crosslinking treatment: Prepare a 5% glutaraldehyde solution, that is, 2 mL of glutaraldehyde (50% w / v) and 20 mL of sterile water; place the prepared solution at the bottom of the desiccator, place the amniotic membrane after dry treatment in the upper part of the desiccator, seal it and place it in the incubator, react at 45 °C for 24 hours, and turn it over every 12 hours; thus, the crosslinking treatment of the amniotic membrane is completed; (6)Dry treatment after amniotic membrane crosslinking: This step is the same as step (4) to obtain a dry amniotic membrane with non-freeze-dried steam crosslinking.

[0046] Test Example 1 Uniaxial tensile mechanical test In this test example, the above Examples 1-6 and Comparative Examples 1-11 were subjected to uniaxial tensile mechanical tests, and the specific process is as follows: Cut the examples and control examples into five groups of rectangles with a size of 0.5×2 cm respectively, fix both ends of the samples on the upper and lower clamps of a tensile tester (NK-10 pointer push-pull gauge, manufacturer: AIGU) as Figure 3 shown, measure the width of the sample with a ruler, and test the thickness of the sample with a thickness gauge (0-12.7 mm flat head micrometer, manufacturer: Everite) as Figure 2 shown, turn the knob on the tensile tester to "Peak", slowly and evenly shake the tensile tester until the sample breaks, record the maximum tensile force for breaking the sample, and calculate the corresponding stress. The results are shown in Table 1 and Figure 6 shown.

[0047] It should be noted that Examples 1-6 are dry amniotic membranes with non-freeze-dried steam crosslinking obtained by the preparation method described in the present invention. Comparative Example 1 is a fresh amniotic membrane without treatment. Comparative Example 2 is an uncrosslinked amniotic membrane air-dried naturally. Comparative Example 3 is an uncrosslinked fresh amniotic membrane after being atomized and sprayed with the hydrophilic hydrogen bond supplement reagent in the present invention. Comparative Example 4 is an amniotic membrane obtained by using a very low concentration glutaraldehyde crosslinking agent in the preparation method described in the present invention. Comparative Example 5 is an amniotic membrane crosslinked by soaking in a 1% (w / v) glutaraldehyde crosslinking agent. Comparative Example 6 is an amniotic membrane obtained by freeze-drying the steam crosslinking preparation method described in the present invention. Comparative Example 7 is an amniotic membrane obtained by air-drying the steam crosslinking preparation method described in the present invention. Comparative Example 8 is a dry amniotic membrane treated with 5% glutaraldehyde crosslinking agent by steam crosslinking and only treated with the hydrogen bond supplement reagent before crosslinking. Comparative Example 9 is a dry amniotic membrane treated with 5% glutaraldehyde crosslinking agent by steam crosslinking and only treated with the hydrogen bond supplement reagent after crosslinking. Comparative Example 10 is a dry amniotic membrane treated with 5% glutaraldehyde crosslinking agent by steam crosslinking and treated with a high concentration hydrogen bond supplement reagent in the dry state. Comparative Example 11 is a dry amniotic membrane treated with 5% glutaraldehyde crosslinking agent by steam crosslinking and treated with a low concentration hydrogen bond supplement reagent in the dry state.

[0048] Table 1 As can be seen from Table 1 and Figure 6 the results: (1) For the amniotic membranes of Examples 1-3 prepared by the method of the present invention, compared with the uncrosslinked amniotic membrane treated with a hydrogen bond replenishing reagent in Comparative Example 3, the maximum tensile fracture stress has increased. It has increased from 0.61 MPa in Comparative Example 3 to 2.64, 3.85, and 4.21 MPa respectively, that is, an increase of 332.79%, 531.1%, and 590.2%. It can be seen that the steam-crosslinked amniotic membrane prepared by the method of the present invention has significantly improved mechanical strength compared with the uncrosslinked amniotic membrane, achieving an important purpose in the content of the present invention.

[0049] (2) For the amniotic membranes of Examples 1, 4-6 prepared by the method of the present invention, compared with the freeze-dried crosslinked amniotic membrane in Comparative Example 6 and the air-dried crosslinked amniotic membrane in Comparative Example 7, the maximum tensile fracture stress has increased by 0.99-3.02 MPa. Among them, the relatively excellent Example 2 has increased the maximum tensile fracture stress by 101.64% and 121.85% compared with the amniotic membranes of Comparative Example 6 and Comparative Example 7. It can be seen that the crosslinked amniotic membrane treated with a hydrophilic hydrogen bond replenishing reagent before and after crosslinking in the method of the present invention has greatly improved mechanical strength compared with the freeze-dried and air-dried amniotic membranes. The maximum tensile fracture stress of the fresh amniotic membrane treated with the hydrophilic hydrogen bond replenishing reagent described in the present invention in Comparative Example 3 is similar to that of the fresh amniotic membrane in Comparative Example 1. It can be seen that the amniotic membrane treated with the hydrophilic hydrogen bond replenishing reagent described in the present invention can maintain mechanical strength comparable to that of the fresh amniotic membrane. In Comparative Examples 8 and 9, only before or after crosslinking, the hydrophilic hydrogen bond replenishing reagent was used to compare with the air-dried amniotic membrane, and the improvement was 27.73% and 1.68%. It can be seen that treating with a hydrophilic hydrogen bond replenishing reagent before and after crosslinking has a significant improvement in mechanical strength compared with treating with a hydrophilic hydrogen bond replenishing reagent only before or only after crosslinking once.

[0050] (3) For the amniotic membrane of Example 6 prepared by the method of the present invention, compared with the very low concentration of hyaluronic acid (hydrogen bond replenishing reagent) in Comparative Example 11, the maximum tensile fracture stress has increased by 1.07 Mpa. It can be seen that the concentration range of the hydrogen bond replenishing reagent is an important value to ensure the dry-state treatment result. Too low a concentration cannot achieve the dry-state effect and thus affects the mechanical strength. There is little difference compared with the high concentration of hyaluronic acid in Comparative Example 10. It can be seen that treatment above this concentration range (0.5-2.0%) has little significance and high-concentration treatment is not required.

[0051] (4)The amniotic membranes of Examples 1 to 3 prepared by the method of the present invention, compared with the amniotic membrane crosslinked with extremely low-concentration steam in Comparative Example 4 using the method of the present invention, the maximum tensile fracture stress increased by 1.34 to 3.34 MPa, and the fracture stress of the better Example 3 compared with that of the amniotic membrane in Comparative Example 4 increased by 383.9%. It can be seen that the selection of the crosslinking agent concentration in the present invention is an important crosslinking condition, and the concentrations in the better examples of the present invention are all appropriate concentrations, that is, they can effectively improve the mechanical strength of the amniotic membrane.

[0052] Test Example 2 Collagenase degradation test The above Examples 1-6 and Comparative Examples 1-11 were subjected to collagenase degradation tests, and the specific process was as follows: Cut the examples and control examples into squares with a size of 1 cm×1 cm, with three groups each. Place the samples in a drying oven to dry, and weigh the dried samples with a balance and record it as W0; after the above steps are completed, place the samples in 1.5-ml centrifuge tubes, and then add 1.5 ml of a mixed solution of 1% pepsin and 0.1 M hydrochloric acid to them, place them in an incubator at 37°C for 24 hours, take out the enzymatically digested samples, dry and weigh them and record it as W1. The comparison results before and after the experiment are as Figure 4 、 Figure 5 。The weight loss rate of the samples was calculated using the following formula to characterize the enzymatic degradation resistance of the materials: Sample weight loss rate = (W0 - W1) / W0×100%.

[0053] The results are as shown in Table 2 and Figure 7 shown.

[0054] Table 2 From Table 2 and Figure 7 the results, it can be seen that: (1)The amniotic membranes of the better Examples 1 to 6 prepared by the method of the present invention, compared with the uncrosslinked amniotic membranes in Comparative Examples 1 to 3, the enzymatic degradation weight loss rate decreased by 32.68% to 20.33%. It can be seen that the steam-crosslinked amniotic membrane of the present invention can effectively reduce the degradation rate compared with the uncrosslinked amniotic membrane, and its anti-protease degradation performance is improved.

[0055] (2)The weight loss rate after enzymatic degradation of the amniotic membranes of the better Examples 1 to 6 prepared by the method of the present invention is equivalent to that of the amniotic membranes crosslinked by soaking in Comparative Example 5, indicating that the steam crosslinking of the present invention can achieve the effectiveness of soaking crosslinking.

[0056] (3)Compared with the extremely low-concentration steam-crosslinked amniotic membrane in Comparative Example 4, the weight loss rate of the amniotic membrane in the preferred Example 1 prepared by the method of the present invention was effectively reduced by 28.35%, indicating that the selection of the crosslinking agent concentration in the present invention is an important crosslinking condition. The concentrations in the examples of the present invention are all appropriate concentrations, that is, they can effectively reduce the weight loss rate after enzymatic degradation, and its anti-protease degradation performance is improved.

[0057] The embodiments of the present application have been described above in conjunction with the accompanying drawings. Specific examples have been used in this article to elaborate on the principles and implementation manners of the present application. The descriptions of the above embodiments are only used to help understand the core idea of the present application. However, the present application is not limited to the above specific implementation manners. The above specific implementation manners are only illustrative and not restrictive. Under the inspiration of the present application, those of ordinary skill in the art can also make many forms without departing from the purpose of the present application and the scope protected by the claims, and all belong to the protection scope of the present application.

Claims

1. A method for preparing dry amniotic membrane based on steam cross-linking, characterized in that: The following steps are involved: S1, using a hydrophilic hydrogen bond supplement reagent to spray the pretreated amniotic membrane sample, and let it stand; S2, steam cross-linking the standing amniotic membrane sample using a cross-linking agent; S3, atomizing and spraying the steam-crosslinked amniotic membrane sample again with a hydrophilic hydrogen bond supplementing reagent, and letting it stand to obtain the dry amniotic membrane; The hydrophilic hydrogen bond supplementing agent includes one or more of a compound containing an oxygen group and a compound containing a nitrogen group; The cross-linking agent includes glutaraldehyde solution.

2. The method according to claim 1, characterized in that: The pre-processing comprises: The fresh amniotic membrane was washed with water to remove surface impurities and then fixed.

3. The method according to claim 1, characterized in that The specific steps of the steam cross-linking are as follows: The cross-linking agent is placed in a desiccator, and the amniotic membrane sample is placed 5 cm to 15 cm above the cross-linking agent for 24 to 48 hours, and the amniotic membrane sample is turned over every 12 hours or 24 hours.

4. The method according to claim 1, characterized in that: The hydrophilic hydrogen bond supplementing reagent includes one or more of glycerol, ethanol, tyrosine solution, carboxymethyl chitosan solution, and hyaluronic acid solution.

5. The method according to claim 4, characterized in that The hydrophilic hydrogen bond supplementing reagent is a mixed solution of glycerol and ethanol.

6. The method according to claim 5, characterized in that The volume ratio of the glycerol to the ethanol is 2:

8.

7. The method according to claim 4, characterized in that The concentration of the tyrosine solution is 0.5% to 2%, the concentration of the carboxymethyl chitosan solution is 0.5% to 5%, and the concentration of the hyaluronic acid solution is 0.5% to 2%.

8. The method according to claim 1, characterized in that: The concentration of the glutaraldehyde solution is 5% to 25%.

9. A dry amniotic membrane, characterized in that: The method is prepared by any one of claims 1 to 8.

10. Use of the dry amniotic membrane as claimed in claim 9 in the preparation of materials for promoting tissue repair and regeneration.

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