Performance-enhanced biological amnion and preparation method thereof

Abstract

The invention provides a performance-enhanced biological amniotic membrane and a preparation method thereof, and the preparation method comprises the following steps: S1, swelling: spreading a single-layer amniotic membrane on a support which is pre-infiltrated with an acid solution, and enabling one side of a sponge layer of the amniotic membrane to be in full contact with the support; s2, bonding: fully soaking the sample prepared in the step S1 in a bonding solution, taking out the amnion, spreading the amnion, and sequentially spreading a plurality of layers of amnions soaked in the bonding solution on the spread amnion; s3, dehydration: carrying out dehydration treatment on the sample prepared in S2; s4, crosslinking is carried out; the biological amniotic membrane is formed by bonding the multiple layers of amniotic membranes through the bonding solution containing glycerin, the thickness of the product is greatly increased, the multiple layers of amniotic membranes can still be firmly bonded together after the product is rehydrated, the layering phenomenon is avoided, a novel photo-crosslinking technology is adopted, the degradation speed of the biological amniotic membrane is lower, and the biological amniotic membrane is suitable for wounds with certain inflammatory response; and the situation that the biological amnion is dissolved too early and loses the wound barrier effect can be avoided.

Description

Technical Field

[0001] The present invention belongs to the category of medical products, and relates to a performance-enhanced biological amniotic membrane and a preparation method thereof. The product can be used in the fields of ophthalmology, dermatology, trauma orthopedics, and is mainly used for the regeneration and repair of soft tissue injuries. Background Art

[0002] The amniotic membrane of mammals is a semi-transparent membrane on the innermost side of the placenta. It has no blood vessels, nerves or lymph, and has a certain degree of elasticity and toughness. Human amniotic membrane can be processed by bioengineering technology to produce an allogeneic medical device "bio-amniotic membrane", which has multiple biological activities, such as: excellent biocompatibility, almost no immune rejection reaction; antiviral and bactericidal effects, and a low risk of infection after transplantation; inhibiting local inflammatory reactions and preventing new blood vessels and scar formation. At present, bio-amniotic membrane has been widely used in ophthalmic diseases such as pterygium, corneal ulcers, chemical burns, and blepharobulbar adhesions. It can also be used to promote the regeneration and repair of tendons, peripheral nerves, and periosteum, as well as burn surgery, plastic surgery, gynecology, urology, etc. to reconstruct mucosal and skin tissues.

[0003] The total thickness of the amniotic membrane is 0.02mm-0.05m. It has a multi-layer structure, which is divided into the epithelial layer, basement membrane layer, matrix layer, fibroblast layer and sponge layer from the inside to the outside. Since the blood-contaminated part needs to be removed during the preparation process, the biological amniotic membrane product is thinner than the fresh amniotic membrane. It is generally attached to a nitrocellulose film (NC membrane) and peeled off before clinical use. However, the thin and soft nature of the biological amniotic membrane makes it very easy to shrink into a ball after being peeled off from the NC membrane, which is not convenient for subsequent clinical operations such as suturing. In addition, the clinical application scenarios of biological amniotic membranes often involve wounds with certain inflammatory reactions, and the biological amniotic membrane covering them is very easy to dissolve prematurely, thereby losing its wound barrier function. Therefore, it is urgent to develop a performance-enhanced biological amniotic membrane and its preparation method to solve the above technical problems.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The purpose of the present invention is to provide a performance-enhanced biological amniotic membrane and a preparation method thereof. The biological amniotic membrane is formed by bonding multiple layers of amniotic membranes through an adhesive solution containing glycerol. The thickness of the product is greatly increased, which solves the problem of inconvenience in suturing caused by the amniotic membrane being too thin and too soft. After the product is rehydrated, the multiple layers of amniotic membranes can still be firmly adhered together to avoid stratification. A new type of photo-crosslinking process is used to slow down the degradation rate of the biological amniotic membrane. The biological amniotic membrane is suitable for wounds with certain inflammatory reactions, and can avoid premature dissolution of the biological amniotic membrane and loss of its wound barrier function.

[0006] In order to achieve the above-mentioned object, the present invention provides a method for preparing a performance-enhanced biological amniotic membrane, comprising the following steps: S1: Swelling: Spread a single layer of amniotic membrane on a support pre-soaked in an acidic solution, so that the sponge layer side of the amniotic membrane is in full contact with the support; S2: bonding, placing the sample prepared in S1 in a bonding solution and fully soaking it, taking out the amniotic membrane, laying it flat, and laying multiple layers of amniotic membrane soaked in the bonding solution on the flattened amniotic membrane in sequence, wherein the bonding solution comprises glycerol, a bonding agent and water, wherein the mass fraction of glycerol is 1-90%, the mass fraction of the bonding agent is 0-10%, and the bonding agent is gelatin, hyaluronic acid or a mixture of the two; S3: dehydration, dehydrating the sample prepared in S2 until the water is completely evaporated and the sample becomes translucent; S4: cross-linking, the sample prepared in S3 is fully immersed in a photo-cross-linking solution, taken out and flattened, and photo-cross-linked by ultraviolet light or blue light irradiation, wherein the photo-cross-linking solution comprises glycerol, riboflavin substances and some water, wherein the mass fraction of glycerol is 30-90%, and the mass fraction of riboflavin substances is 0.1-3%.

[0007] Preferably, in S1 and S2, the amniotic membrane is natural amniotic membrane or amniotic membrane that has been decellularized, and the amniotic membrane is derived from the placenta of a mammal, and the mammal is a human, pig, sheep, horse or cow.

[0008] Preferably, in S1, the acidic solution is an organic acid solution with a mass fraction of 0.01-3%, the organic acid is formic acid, acetic acid, citric acid, tartaric acid, malic acid or ascorbic acid, and the support is filter paper, non-woven fabric or gauze.

[0009] Preferably, in S3, the dehydration treatment is natural air drying, oven drying, vacuum drying or vacuum freeze drying.

[0010] Preferably, in S4, the riboflavin substance is riboflavin or a riboflavin derivative.

[0011] Preferably, in S4, the irradiation intensity of the photocrosslinking is 1-30 mW / cm 2 , irradiation dose is 1.0 -30.0 J / cm 2 .

[0012] Preferably, the sample prepared in S4 is directly sterilized by irradiation without dehydration or sterilized by irradiation after dehydration or sterilized by ethylene oxide after dehydration.

[0013] The present invention also provides a performance-enhanced biological amniotic membrane prepared by the preparation method of the performance-enhanced biological amniotic membrane.

[0014] The present invention provides a performance-enhanced biological amniotic membrane and a preparation method thereof, which have the following beneficial effects.

[0015] 1. The present invention is made of multiple layers of amniotic membrane, the thickness of the product is greatly increased, and the problem of inconvenience in suturing caused by the amniotic membrane being too thin and too soft is solved.

[0016] 2. The production process of the present invention is simple, and after the product is rehydrated, the multiple layers of amniotic membrane can still be firmly adhered together to avoid stratification.

[0017] 3. The present invention adopts a novel photo-crosslinking process, which makes the degradation rate of the biological amnion slower, and is suitable for wounds with certain inflammatory reactions, and can avoid premature dissolution of the biological amnion and loss of its wound barrier function. DETAILED DESCRIPTION

[0018] The present invention is further described below in conjunction with specific embodiments to help understand the content of the present invention.

[0019] The present invention provides a method for preparing a performance-enhanced biological amniotic membrane, comprising the following steps: S1: Swelling, spreading a single layer of amniotic membrane on a support pre-soaked in an acidic solution, so that the sponge layer side of the amniotic membrane is in full contact with the support; the acidic solution is an organic acid solution with a mass fraction of 0.01-3%, and the organic acid is formic acid, acetic acid, citric acid, tartaric acid, malic acid or ascorbic acid, and the support is filter paper, non-woven fabric or gauze.

[0020] S2: bonding, placing the sample prepared in S1 in a bonding solution and fully soaking it, taking out the amniotic membrane and flattening it, and sequentially laying multiple layers of amniotic membrane soaked in the bonding solution on the flattened amniotic membrane, wherein the bonding solution comprises glycerol, an adhesive and water, wherein the mass fraction of glycerol is 1-90%, the mass fraction of the adhesive is 0-10%, and the adhesive is gelatin, hyaluronic acid or a mixture of the two.

[0021] In S1 and S2, the amniotic membrane is natural amniotic membrane or amniotic membrane that has been decellularized, and the amniotic membrane is derived from the placenta of a mammal, and the mammal is a human, pig, sheep, horse or cow.

[0022] S3: Dehydration, dehydrating the sample prepared in S2 until the water is completely evaporated and the sample becomes translucent, wherein the dehydration treatment is natural air drying, oven drying, vacuum drying or vacuum freeze drying.

[0023] S4: Cross-linking, the sample prepared in S3 is placed in a photo-cross-linking solution and fully immersed, taken out and laid flat, and photo-cross-linked by ultraviolet light or blue light irradiation, wherein the photo-cross-linking solution includes glycerol, riboflavin substances and some water, wherein the mass fraction of glycerol is 30-90%, and the mass fraction of riboflavin substances is 0.1-3%. The riboflavin substances are riboflavin or riboflavin derivatives. The irradiation intensity of the photo-cross-linking is 1-30 mW / cm 2, irradiation dose is 1.0 -30.0 J / cm 2 The sample prepared in S4 is directly sterilized by irradiation without dehydration or sterilized by irradiation after dehydration or sterilized by ethylene oxide after dehydration.

[0024] The present invention also provides a performance-enhanced biological amniotic membrane prepared by the preparation method of the performance-enhanced biological amniotic membrane.

[0025] The experimental methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents, etc. used can be purchased from commercial channels unless otherwise specified; the preparation method of the single-layer amniotic membrane or single-layer decellularized amniotic membrane described in the present invention is based on the conventional methods mentioned in academic literature.

[0026] Example 1: A method for preparing human-derived biological amniotic membrane According to the conventional method, human placenta was obtained, the amniotic membrane was peeled off and the blood stains were cleaned to obtain a single layer of amniotic membrane, and then the multi-layer composite amniotic membrane was prepared by the following steps: S1: Swelling: Take a piece of filter paper and soak it fully in 0.1% (mass fraction) acetic acid solution. Spread a single layer of amniotic membrane on the filter paper so that the sponge layer of the amniotic membrane is in full contact with the filter paper. Take it out after 15 minutes. S2: bonding, the sample prepared in the previous step is placed in a bonding solution and fully soaked, the amniotic membrane is taken out and laid flat, and in order to prepare a double-layer composite amniotic membrane, another layer of amniotic membrane soaked in the bonding solution is laid on the flattened amniotic membrane. The bonding solution is prepared according to the following formula (mass fraction): Glycerin 50% Water 50% S3: Dehydration: Place the sample prepared in the previous step into a vacuum dryer for vacuum drying until the water is completely evaporated and the sample becomes translucent; S4: Cross-linking: the sample prepared in the previous step was immersed in a photo-cross-linking solution for 30 minutes, taken out and laid flat, and irradiated with 365 nm ultraviolet light for photo-cross-linking, wherein the ultraviolet irradiation intensity was 10 mW / cm 2 The irradiation dose was 5.4 J / cm 2 The photo-crosslinking solution is prepared according to the following formula (mass fraction): Glycerin 50% Riboflavin 1% Water 49% The cross-linked sample in the previous step is rinsed until no obvious yellow color is present, and then directly irradiated and sterilized after packaging to obtain a wet biological amniotic membrane product.

[0027] Example 2: A method for preparing a porcine decellularized biological amniotic membrane First, the porcine placenta was taken, the amniotic membrane was removed to clean the blood stains, and a single layer of porcine decellularized biological amniotic membrane was prepared by conventional decellularization methods in the literature, and then a multilayer composite amniotic membrane was prepared by the following steps: S1: Swelling: Take a piece of non-woven fabric and soak it fully in 0.03% (mass fraction) formic acid solution. Spread a single layer of amniotic membrane on the non-woven fabric so that the sponge layer of the amniotic membrane is in full contact with the non-woven fabric. Take it out after 30 minutes. S2: bonding, the sample prepared in the previous step is fully soaked in the bonding solution, the amniotic membrane is taken out and laid flat, and in order to prepare a three-layer composite amniotic membrane, two layers of amniotic membrane soaked in the bonding solution are laid on the flat amniotic membrane in sequence. The bonding solution is prepared according to the following formula (mass fraction): Glycerin 1% Gelatin 10% Water 89% S3: Dehydration: Place the sample prepared in the previous step in an oven for drying until the water is completely evaporated and the sample becomes translucent; S4: Cross-linking: the sample prepared in the previous step was immersed in a photo-cross-linking solution for 60 minutes, taken out and laid flat, and irradiated with 254 nm ultraviolet light for photo-cross-linking, wherein the ultraviolet irradiation intensity was 1 mW / cm 2 , the irradiation dose is 1.0 J / cm 2 The photo-crosslinking solution is prepared according to the following formula (mass fraction): Glycerin 30% Riboflavin 0.1% Water 69.9% The cross-linked sample in the previous step is rinsed until no obvious yellow color is present, and then sterilized by irradiation after dehydration and drying to obtain a dry biological amniotic membrane product. The dehydration and drying can be performed by air drying, oven drying, vacuum drying, vacuum freeze drying or organic solvent dehydration and drying.

[0028] Example 3: A method for preparing bovine decellularized biological amniotic membrane First, a bovine placenta was obtained, the amniotic membrane was removed to clean the blood stains, and a single layer of bovine decellularized biological amniotic membrane was prepared using a conventional decellularization method in the literature. Then, a multilayer composite amniotic membrane was prepared using the following steps: S1: Swelling: Take a piece of gauze and soak it fully in 3% (mass fraction) citric acid solution. Spread a single layer of amniotic membrane on the gauze so that the sponge layer of the amniotic membrane is in full contact with the gauze. Take it out after 5 minutes. S2: bonding, the sample prepared in the previous step is placed in a bonding solution and fully soaked, the amniotic membrane is taken out and laid flat, and in order to prepare a five-layer composite amniotic membrane, four layers of amniotic membrane soaked in the bonding solution are laid on the flattened amniotic membrane in sequence. The bonding solution is prepared according to the following formula (mass fraction): Glycerin 90% Hyaluronic acid 1% Water 9% S3: Dehydration: air-dry the sample prepared in the previous step until the water is completely evaporated and the sample becomes translucent; S4: Cross-linking: the sample prepared in the previous step was immersed in the photo-cross-linking solution for 30 minutes, taken out and flattened, and photo-cross-linked by blue light irradiation, wherein the irradiation intensity was 30 mW / cm 2 , the irradiation dose is 30.0 J / cm 2 The photo-crosslinking solution is prepared according to the following formula (mass fraction): Glycerin 90% Riboflavin Phosphate 3% Water 7% The cross-linked sample in the previous step is rinsed until no obvious yellow color is present, and then sterilized with ethylene oxide to obtain a dry biological amniotic membrane product. The dehydration and drying can be performed by air drying, oven drying, vacuum drying, vacuum freeze drying or organic solvent dehydration and drying.

[0029] Comparative Example 1: Preparation and performance comparison test of single-layer biological amniotic membrane According to the method described in Example 1, a human placenta was obtained, the amniotic membrane was peeled off and the blood stains were cleaned to obtain a single-layer amniotic membrane, which was then directly irradiated and sterilized to obtain a wet single-layer biological amniotic membrane. Then, the thickness and suturing convenience of the wet biological amniotic membranes prepared in Comparative Example 1 and Examples 1-3 were tested according to the following method.

[0030] Thickness test: Take 3 pieces of biological amniotic membrane samples prepared in Example 1 and Comparative Example 1, and use a thickness gauge to measure the sample thickness; the experimental results show that the thickness of the comparative example 1 sample is 14±3 microns, the thickness of the example 1 sample is 37±5 microns, the thickness of the example 2 sample is 64±6 microns, and the thickness of the example 3 sample is 91±7 microns.

[0031] Suture convenience test: Take the samples after the above thickness test and use 6-0 suture thread (with needle) to perform suture test. The results show that compared with the sample of comparative example 1 which is easy to curl up, the samples of examples 1-3 are easier to be clamped with tweezers and sutured.

[0032] The above tests show that compared with the biological amniotic membrane prepared by conventional processes, the thickness of the product of the present invention is greatly increased, which solves the problem of inconvenient suturing caused by the amniotic membrane being too thin and too soft.

[0033] Comparative Example 2: Preparation of conventional multi-layer biological amniotic membrane and its performance comparison test Except for deleting step S4 in Example 2, the remaining steps were carried out according to the method described in Example 2 to prepare a conventional multi-layer composite amniotic membrane. Then, the adhesion firmness and degradation performance of the multi-layer composite amniotic membrane prepared in Example 2 and Comparative Example 2 were tested according to the following method.

[0034] Adhesion firmness test: 10 samples prepared in Example 2 and Comparative Example 2 were taken, immersed in biological saline at 37°C for 72 hours, and observed for stratification; then the samples were shaken at 100 rpm for 72 hours and observed for stratification again. The experimental results showed that 3 samples prepared in Comparative Example 2 were stratified after being left to stand for 72 hours, and all of them were stratified after being shaken at 100 rpm for 72 hours; while stratification occurred from beginning to end in Example 2.

[0035] Degradation performance test: 12 rabbits were taken, and the animals were conventionally anesthetized before surgery. A monocular corneal alkali burn model was prepared according to the literature method. The biological amniotic membrane prepared in Example 2 or Comparative Example 2 was cut to an appropriate size, and then 6 animals were selected, and the amniotic membrane was covered on the corneal wound site and sutured conventionally. The dissolution of the amniotic membrane was generally observed 3 days, 7 days, and 14 days after surgery. The experimental results showed that one animal in the comparative example 2 sample was completely dissolved 3 days after surgery (accounting for 50%), and all dissolved 7 days after surgery; while only one piece of the sample in Example 2 was partially dissolved 7 days after surgery, and only 2 pieces were not completely dissolved 14 days after surgery. Moisten the amniotic membrane with physiological saline and gently peel it off, and it can be observed that the site of corneal alkali burn has been completely re-epithelialized.

[0036] The above tests show that compared with the multi-layer composite amniotic membrane prepared without the photocrosslinking process, the multi-layer amniotic membrane of the product of the present invention can still be firmly adhered together after rehydration to avoid stratification. Animal experiments on rabbit corneal alkali burns show that the degradation rate of the product of the present invention is slower, and it is suitable for wounds with certain inflammatory reactions, and can avoid premature dissolution of the biological amniotic membrane and loss of its wound barrier function.

[0037] The invention concept is described in detail using specific examples herein, and the description of the above embodiments is only used to help understand the core idea of ​​the invention. It should be pointed out that any obvious modification, equivalent substitution or other improvement made by a person of ordinary skill in the art without departing from the invention concept should be included in the protection scope of the present invention.

Claims

1. A method for preparing a performance-enhanced biological amniotic membrane, characterized in that: The steps include: S1: Swelling: Spread a single layer of amniotic membrane on a support pre-soaked in an acidic solution, so that the sponge layer side of the amniotic membrane is in full contact with the support; S2: bonding, placing the sample prepared in S1 in a bonding solution and fully soaking it, taking out the amniotic membrane, laying it flat, and laying multiple layers of amniotic membrane soaked in the bonding solution on the flattened amniotic membrane in sequence, wherein the bonding solution comprises glycerol, a bonding agent and water, wherein the mass fraction of glycerol is 1-90%, the mass fraction of the bonding agent is 0-10%, and the bonding agent is gelatin, hyaluronic acid or a mixture of the two; S3: dehydration, dehydrating the sample prepared in S2 until the water is completely evaporated and the sample becomes translucent; S4: cross-linking, the sample prepared in S3 is fully immersed in a photo-cross-linking solution, taken out and flattened, and photo-cross-linked by ultraviolet light or blue light irradiation, wherein the photo-cross-linking solution comprises glycerol, riboflavin substances and some water, wherein the mass fraction of glycerol is 30-90%, and the mass fraction of riboflavin substances is 0.1-3%.

2. The method for preparing a performance-enhanced biological amniotic membrane according to claim 1, characterized in that: In S1 and S2, the amniotic membrane is natural amniotic membrane or amniotic membrane that has been decellularized, and the amniotic membrane is derived from the placenta of a mammal, and the mammal is a human, pig, sheep, horse or cow.

3. The method for preparing a performance-enhanced biological amniotic membrane according to claim 2, characterized in that: In S1, the acidic solution is an organic acid solution with a mass fraction of 0.01-3%, the organic acid is formic acid, acetic acid, citric acid, tartaric acid, malic acid or ascorbic acid, and the support is filter paper, non-woven fabric or gauze.

4. The method for preparing a performance-enhanced biological amniotic membrane according to claim 3, characterized in that: In S3, the dehydration treatment is natural air drying, oven drying, vacuum drying or vacuum freeze drying.

5. The method for preparing a performance-enhanced biological amniotic membrane according to claim 4, characterized in that: In S4, the riboflavin substance is riboflavin or a riboflavin derivative.

6. The method for preparing a performance-enhanced biological amniotic membrane according to claim 5, characterized in that: In S4, the irradiation intensity of the photocrosslinking is 1-30 mW / cm 2 , irradiation dose is 1.0 -30.0 J / cm 2 .

7. The method for preparing a performance-enhanced biological amniotic membrane according to claim 6, characterized in that: The sample prepared in S4 is directly sterilized by irradiation without dehydration or sterilized by irradiation after dehydration or sterilized by ethylene oxide after dehydration.

8. A performance-enhanced biological amniotic membrane prepared by the method for preparing a performance-enhanced biological amniotic membrane according to any one of claims 1 to 7.