Performance-enhanced biological amniotic membrane and method of making same

Abstract

The application provides a performance-enhanced biological amniotic membrane and a preparation method thereof, and comprises the following steps: S1: swelling, laying a single-layer amniotic membrane on a support which is previously soaked with an acidic solution, and making the sponge layer of the amniotic membrane fully contact with the support; S2: bonding, soaking the sample prepared in S1 in a bonding solution, taking out the amniotic membrane, laying it flat, and laying multiple layers of amniotic membranes which are soaked in the bonding solution on the laid amniotic membrane; S3: dehydration, carrying out a dehydration treatment on the sample prepared in S2; and S4: cross-linking; the multiple layers of amniotic membranes are bonded by the bonding solution containing glycerol, the thickness of the product is greatly increased, the multiple layers of amniotic membranes can still be firmly adhered together after rehydration of the product, and the delamination phenomenon is avoided, a novel photo-crosslinking process is adopted, the degradation speed of the biological amniotic membrane is slower, the biological amniotic membrane is suitable for wounds with certain inflammatory reactions, and the biological amniotic membrane can avoid premature dissolution and loss of the wound barrier effect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field 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, etc., and is mainly used for the regeneration and repair of soft tissue injury. BACKGROUND

[0002] The amnion of mammals is the innermost layer of the placenta, which is a semi-transparent thin film without blood vessels, nerves and lymph, and has certain elasticity and toughness. The human amniotic membrane can be processed by biological engineering technology to obtain an allogeneic medical device "biological amniotic membrane", which has multiple biological activities, such as excellent biocompatibility, almost no immune rejection reaction, anti-virus and sterilization effect, less risk of infection after transplantation, inhibition of local inflammatory reaction, and prevention of new blood vessels and scar formation. At present, the biological amniotic membrane has been widely used in pterygium, corneal ulcer, chemical burn, and symblepharon of ophthalmic diseases. It can also be used to promote the regeneration and repair of tendons, peripheral nerves, and periosteum, and to reconstruct mucosa and skin tissue in burn surgery, plastic surgery, gynecology, and urology.

[0003] The total thickness of the amniotic membrane is 0.02mm-0.05m, which has a multi-layer structure, and is divided into an epithelial layer, a basement membrane layer, a matrix layer, a fibroblast layer, and a sponge layer from inside to outside. Since the part contaminated by blood needs to be removed during preparation, the biological amniotic membrane product is thinner than the fresh amniotic membrane, and is generally attached to a nitrocellulose membrane (NC membrane) and is removed before clinical use. However, the thin and soft characteristics of the biological amniotic membrane make it prone to shrink into a ball after being removed from the NC membrane, which is inconvenient for subsequent suture and other clinical operations. In addition, the clinical application scenarios of the biological amniotic membrane often involve wounds with certain inflammatory reactions, and the biological amniotic membrane covering the wound is prone to dissolve too early, thereby losing its wound barrier effect. Therefore, it is urgent to develop a performance-enhanced biological amniotic membrane and a preparation method thereof to solve the above technical problems.

[0004] In view of this, the present application is proposed. SUMMARY

[0005] The purpose of the present application is to provide a performance-enhanced biological amniotic membrane and a preparation method thereof, which is bonded by a multi-layer amniotic membrane through an adhesive solution containing glycerol. The thickness of the product is greatly increased, solving the problem of inconvenience in suturing caused by the too thin and soft amniotic membrane. After rehydration, the multi-layer amniotic membrane can still be firmly adhered together to avoid delamination. A new light cross-linking process is adopted, so that the biological amniotic membrane degrades more slowly, is suitable for wounds with certain inflammatory reactions, and can avoid the premature dissolution of the biological amniotic membrane and the loss of the wound barrier effect.

[0006] In order to achieve the above purpose, the present application provides a preparation method of a performance-enhanced biological amniotic membrane, comprising the following steps:

[0007] S1: swelling, laying a single layer of amniotic membrane on a support previously soaked in an acidic solution, so that the sponge layer of the amniotic membrane is in full contact with the support;

[0008] S2: bonding, soaking the sample prepared in S1 in a bonding solution, taking out the amniotic membrane, laying it flat, and then laying multiple layers of amniotic membrane soaked in the bonding solution on the laid amniotic membrane, the bonding solution comprising glycerol, a bonding agent, and water, wherein the mass fraction of glycerol is 1-90%, and the mass fraction of the bonding agent is 0-10%, the bonding agent being gelatin, hyaluronic acid, or a mixture of the two;

[0009] S3: dehydration, dehydrating the sample prepared in S2 until the water is completely evaporated and the sample is translucent;

[0010] S4: crosslinking, soaking the sample prepared in S3 in a photo-crosslinking solution, taking it out and laying it flat, and then irradiating it with ultraviolet light or blue light for photo-crosslinking, the photo-crosslinking solution comprising glycerol, a riboflavin substance, and water, wherein the mass fraction of glycerol is 30-90%, and the mass fraction of the riboflavin substance is 0.1-3%.

[0011] Preferably, in S1 and S2, the amniotic membrane is a natural amniotic membrane or a decellularized amniotic membrane, and the amniotic membrane is derived from the placenta of a mammal, such as a human, a pig, a sheep, a horse, or a cow.

[0012] Preferably, in S1, the acidic solution is an organic acid solution with a mass fraction of 0.01-3%, the organic acid being formic acid, acetic acid, citric acid, tartaric acid, malic acid, or ascorbic acid, and the support being filter paper, non-woven fabric, or gauze.

[0013] Preferably, in S3, the dehydration treatment is natural air drying, oven drying, vacuum drying, or vacuum freeze-drying.

[0014] Preferably, in S4, the riboflavin substance is riboflavin or a riboflavin derivative.

[0015] Preferably, in S4, the irradiation intensity of photo-crosslinking is 1-30 mW / cm 2 , and the irradiation dose is 1.0-30.0 J / cm 2 .

[0016] Preferably, the sample prepared in S4 is directly irradiated for sterilization without dehydration drying, or is subjected to irradiation sterilization after dehydration drying, or is subjected to ethylene oxide sterilization after dehydration drying.

[0017] The application also provides a performance-enhanced biological amniotic membrane prepared by the above method.

[0018] The application provides a performance enhanced biological amniotic membrane and a preparation method thereof.

[0019] 1. The application is composed of multiple layers of amniotic membranes, and the thickness of the product is greatly increased, thereby solving the problem of inconvenience in suturing caused by the thin and soft amniotic membrane.

[0020] 2. The application has a simple production process, and the multiple layers of amniotic membranes can still be firmly adhered together after rehydration, thereby avoiding delamination.

[0021] 3. The application adopts a novel photocrosslinking process, so that the biological amniotic membrane has a slower degradation speed, is suitable for a wound with certain inflammatory reaction, and can avoid premature dissolution of the biological amniotic membrane and loss of the wound barrier effect. DETAILED DESCRIPTION

[0022] The application will be further described below in combination with specific embodiments, so as to help understand the content of the application.

[0023] The application provides a preparation method of a performance enhanced biological amniotic membrane, which comprises the following steps:

[0024] S1: swelling, the single-layer amniotic membrane is laid on a support pre-soaked with 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%, 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.

[0025] S2: adhesion, the sample prepared in S1 is fully soaked in an adhesion solution, the amniotic membrane is taken out, laid flat, and multiple layers of amniotic membranes soaked in the adhesion solution are laid on the laid amniotic membrane in sequence, the adhesion solution comprises glycerol, an adhesive and water, 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.

[0026] In S1 and S2, the amniotic membrane is natural amniotic membrane or amniotic membrane subjected to decellularization treatment, the amniotic membrane is derived from the placenta of a mammal, and the mammal is a human, a pig, a sheep, a horse or a cow.

[0027] S3: dehydration, the sample prepared in S2 is subjected to dehydration treatment until the water is completely evaporated and the sample is translucent, and the dehydration treatment is natural air drying, oven drying, vacuum drying or vacuum freeze drying.

[0028] S4: cross-linking, the sample prepared in S3 is immersed in a photo-crosslinking solution, and then taken out and laid flat, and photo-crosslinking is performed by irradiation with ultraviolet light or blue light, the photo-crosslinking solution comprising glycerol, a riboflavin substance, and water, wherein the mass fraction of glycerol is 30-90%, and the mass fraction of the riboflavin substance is 0.1-3%. The riboflavin substance is riboflavin or a riboflavin derivative. The irradiation intensity of the photo-crosslinking is 1-30 mW / cm 2 , and the irradiation dose is 1.0-30.0 J / cm 2 . The sample prepared in S4 is directly irradiated for sterilization without dehydration and drying, or is subjected to dehydration and drying and then irradiated for sterilization, or is subjected to dehydration and drying and then sterilized with ethylene oxide.

[0029] The application also provides a performance-enhanced biological amniotic membrane prepared by the preparation method of the performance-enhanced biological amniotic membrane.

[0030] The experimental methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are commercially available unless otherwise specified; the single-layer amniotic membrane or single-layer acellular amniotic membrane described in the application is prepared by referring to the conventional methods mentioned in academic literature.

[0031] Example 1: A preparation method of a human biological amniotic membrane

[0032] A human placenta is taken according to a conventional method, and the amniotic membrane is peeled off, washed to remove blood stains, and prepared into a single-layer amniotic membrane, and then a multi-layer composite amniotic membrane is prepared by using the following steps:

[0033] S1: swelling, a piece of filter paper is fully soaked with 0.1% (mass fraction) acetic acid solution, and the single-layer amniotic membrane is laid flat on the filter paper, so that the sponge layer of the amniotic membrane is in full contact with the filter paper, and the amniotic membrane is taken out after 15 minutes;

[0034] S2: bonding, the sample prepared in the previous step is immersed in a bonding solution, and then 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 laid amniotic membrane. The bonding solution is prepared according to the following formula (mass fraction):

[0035] Glycerol 50%

[0036] Water 50%

[0037] S3: dehydration, the sample prepared in the previous step is placed in a vacuum dryer for vacuum drying until the water is completely evaporated and the sample is translucent;

[0038] S4: Cross-linking, the sample prepared in the previous step was immersed in a photo-crosslinking solution for 30 minutes, taken out and laid flat, and photo-crosslinking was performed using 365 nm ultraviolet irradiation, wherein the ultraviolet irradiation intensity was 10 mW / cm 2 , and the irradiation dose was 5.4 J / cm 2 . The photo-crosslinking solution was prepared according to the following formulation (mass fraction):

[0039] Glycerol 50%

[0040] Riboflavin 1%

[0041] Water 49%

[0042] The cross-linked sample of the previous step was rinsed to be free of obvious yellow color, and was directly irradiation sterilized after packaging, thereby obtaining a wet-state biological amniotic membrane product.

[0043] Example 2: A preparation method of a porcine-derived decellularized biological amniotic membrane

[0044] First, a placenta of porcine origin was taken, the amnion was peeled off, washed of blood, and a single-layer porcine-derived decellularized biological amniotic membrane was prepared using a conventional decellularization method in the literature, and then a multi-layer composite amniotic membrane was prepared using the following steps:

[0045] S1: Swelling, a piece of non-woven fabric was fully immersed in 0.03% (mass fraction) formic acid solution, and the single-layer amniotic membrane was laid flat on the non-woven fabric, with the sponge layer side of the amniotic membrane fully contacting the non-woven fabric, and the sample was taken out after 30 minutes;

[0046] S2: Bonding, the sample prepared in the previous step was immersed in a bonding solution for full immersion, and the amniotic membrane was taken out and laid flat. In order to prepare a three-layer composite amniotic membrane, two layers of amniotic membranes soaked in the bonding solution were sequentially laid on the laid amniotic membrane. The bonding solution was prepared according to the following formulation (mass fraction):

[0047] Glycerol 1%

[0048] Gelatin 10%

[0049] Water 89%

[0050] S3: Dehydration, the sample prepared in the previous step was placed in an oven for drying until the water was completely evaporated and the sample was translucent;

[0051] S4: Cross-linking, the sample prepared in the previous step was immersed in a photo-crosslinking solution for 60 minutes, taken out and laid flat, and photo-crosslinking was performed using 254 nm ultraviolet irradiation, wherein the ultraviolet irradiation intensity was 1 mW / cm 2 , and the irradiation dose was 1.0 J / cm 2 . The photo-crosslinking solution was prepared according to the following formulation (mass fraction):

[0052] Glycerin 30%

[0053] Riboflavin 0.1%

[0054] Water 69.9%

[0055] The cross-linked sample of the previous step is rinsed until no obvious yellow color is observed, and then irradiation sterilization is performed after dehydration drying, to obtain a dry biological amniotic membrane product. The dehydration drying can be air drying, oven drying, vacuum drying, vacuum freeze drying, or organic solvent dehydration drying.

[0056] Example 3: A method for preparing bovine-derived decellularized biological amniotic membrane

[0057] First, a bovine-derived placenta is taken, the amniotic membrane is peeled off, washed of blood, and a single-layer bovine-derived decellularized biological amniotic membrane is prepared using a conventional decellularization method in the literature, and then a multi-layer composite amniotic membrane is prepared using the following steps:

[0058] S1: Swelling, a piece of gauze is taken and soaked with 3% (mass fraction) citric acid solution, the single-layer amniotic membrane is laid flat on the gauze, and the sponge layer side of the amniotic membrane is in full contact with the gauze. After 5 minutes, the amniotic membrane is taken out;

[0059] S2: Adhesion, the sample prepared in the previous step is immersed in the adhesion solution, the amniotic membrane is taken out and laid flat. In order to prepare a five-layer composite amniotic membrane, four layers of amniotic membranes soaked in the adhesion solution are sequentially laid on the laid flat amniotic membrane. The adhesion solution is prepared according to the following formula (mass fraction):

[0060] Glycerin 90%

[0061] Hyaluronic acid 1%

[0062] Water 9%

[0063] S3: Dehydration, the sample prepared in the previous step is naturally air-dried until the water is completely evaporated and the sample is translucent;

[0064] S4: Cross-linking, the sample prepared in the previous step is immersed in a photo-crosslinking solution for 30 minutes, taken out and laid flat, and photo-crosslinking is performed using blue light irradiation, wherein the irradiation intensity is 30 mW / cm 2 , and the irradiation dose is 30.0 J / cm 2 . The photo-crosslinking solution is prepared according to the following formula (mass fraction):

[0065] Glycerin 90%

[0066] Riboflavin phosphate 3%

[0067] Water 7%

[0068] The cross-linked sample of the previous step is rinsed to be free of yellow color, dehydrated and dried, and then sterilized by ethylene oxide to obtain the dry biological amniotic membrane product. The dehydration and drying can be air drying, oven drying, vacuum drying, vacuum freeze drying, or organic solvent dehydration drying.

[0069] Preparation of single-layer biological amniotic membrane and comparison test of its performance

[0070] According to the method described in Example 1, human placenta is taken, the amniotic membrane is peeled off, washed to remove blood stains, and single-layer amniotic membrane is prepared. Then, the single-layer biological amniotic membrane is directly sterilized by irradiation to obtain a wet single-layer biological amniotic membrane. Then, the thickness and suture convenience of the wet biological amniotic membrane prepared in Example 1 and Comparative Example 1 are tested according to the following method.

[0071] Thickness test: three samples of biological amniotic membrane prepared in Example 1 and Comparative Example 1 are taken, and the thickness of the samples is measured using a thickness gauge. The experimental results show that the thickness of the sample in Comparative Example 1 is 14±3 microns, the thickness of the sample in Example 1 is 37±5 microns, the thickness of the sample in Example 2 is 64±6 microns, and the thickness of the sample in Example 3 is 91±7 microns.

[0072] Suture convenience test: the samples after the thickness test are taken, and suture test is performed using 6-0 suture thread (with needle). The results show that compared with the sample in Comparative Example 1 which is easy to roll up, the samples in Examples 1-3 are easier to be clamped with tweezers and sutured.

[0073] The above tests show that compared with the biological amniotic membrane prepared by the conventional process, the thickness of the product of the present application is greatly increased, solving the problem of inconvenience in suture caused by too thin and soft amniotic membrane.

[0074] Preparation of conventional multi-layer biological amniotic membrane and comparison test of its performance

[0075] Except for deleting step S4 in Example 2, the remaining steps are performed according to the method described in Example 2 to prepare a conventional multi-layer composite amniotic membrane. Then, the adhesion strength and degradation performance of the multi-layer composite amniotic membrane prepared in Example 2 and Comparative Example 2 are tested according to the following method.

[0076] Adhesion strength test: 10 samples prepared in Example 2 and Comparative Example 2 are taken, and are soaked in biological saline at 37°C. The delamination of the samples is observed after 72h of standing. Then, the delamination of the samples is observed again after 72h of oscillation at 100rpm. The experimental results show that 3 samples prepared in Comparative Example 2 delaminate after 72h of standing, and all delaminate after 72h of oscillation at 100rpm; while the samples in Example 2 delaminate throughout.

[0077] Degradation performance test: 12 rabbits were taken, and the animals were routinely anesthetized before operation. A single 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, then 6 animals were selected for each, and the amniotic membrane was covered on the corneal wound site and routinely sutured. The amniotic membrane dissolution was observed 3 days, 7 days and 14 days after operation. The experimental results show that one animal of the sample of Comparative Example 2 completely dissolved (accounting for 50%) 3 days after operation, and all dissolved 7 days after operation; while the sample of Example 2 only partially dissolved 1 piece 7 days after operation, and only 2 pieces did not completely dissolve 14 days after operation. The amniotic membrane was moistened with normal saline and gently removed, and it was observed that the site of corneal alkali burn had completely re-epithelialized.

[0078] The above tests show that: compared with the multi-layer composite amniotic membrane prepared without using the photo-crosslinking process, the multi-layer amniotic membrane of the product of the present application can still firmly adhere together after rehydration, avoiding delamination; the rabbit corneal alkali burn animal experiment shows that the degradation speed of the product of the present application is slower, which is suitable for wounds with certain inflammatory reaction, and can avoid the premature dissolution of the biological amniotic membrane and the loss of the wound barrier effect.

[0079] Specific examples are applied in this paper to elaborate the inventive concept in detail, and the above examples are only used to help understand the core idea of the present application. It should be pointed out that any obvious modification, equivalent replacement or other improvement made by those skilled in the art without departing from the inventive concept should be included in the protection scope of the present application.

Claims

1. A method of preparing a performance-enhanced biological amniotic membrane, characterized by, The method comprises the following steps: S1: swelling, spreading the single-layer natural amniotic membrane or the amniotic membrane treated by decellularization on the support pre-soaked with the acidic solution, and making the sponge layer of the amniotic membrane fully contact with the support, wherein the acidic solution is an organic acid solution with a mass fraction of 0.01-3%; S2: bonding, soaking the sample prepared in S1 in the bonding solution, taking out the amniotic membrane, spreading, and spreading the amniotic membrane on the amniotic membrane, wherein the bonding solution comprises glycerol, adhesive and water, the mass fraction of glycerol is 1-90%, the mass fraction of adhesive is 0-10%, and the adhesive is gelatin or a mixture of gelatin and hyaluronic acid; S3: dehydration, dehydrating the sample prepared in S2 until the water is completely evaporated and the sample is translucent; S4: crosslinking, the sample prepared in S3 is immersed in a light crosslinking solution, taken out and laid flat, and light crosslinking is performed by irradiation with ultraviolet light or blue light, the light crosslinking solution comprising glycerol, a riboflavin substance and water, wherein the mass fraction of glycerol is 30-90%, the mass fraction of the riboflavin substance is 0.1-3%, the irradiation intensity of light crosslinking is 1-30 mW / cm 2 , and the irradiation dose is 1.0-30.0 J / cm 2 .

2. The method for preparing a performance-enhanced biological amnion according to claim 1, characterized in that, In S1 and S2, the amniotic membrane is derived from the placenta of mammals, and the mammals are humans, pigs, sheep, horses or cattle.

3. The method for preparing a performance-enhanced biological amnion according to claim 2, characterized in that, In S1, 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 amnion 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 amnion according to claim 4, characterized in that, In S4, the riboflavin substance is riboflavin or riboflavin derivative.

6. The method for preparing a performance-enhanced biological amnion according to claim 5, characterized in that, The sample prepared in S4 is directly sterilized by irradiation without dehydration and drying, or is sterilized by irradiation after dehydration and drying, or is sterilized by ethylene oxide after dehydration and drying.

7. A performance-enhanced biological amniotic membrane prepared by the method for preparing the performance-enhanced biological amniotic membrane according to any one of claims 1-6.