A composite bioactive membrane material, a preparation method and application thereof

Abstract

The application belongs to the technical field of medical materials, and particularly relates to a composite bioactive film material and a preparation method and application thereof, which comprises a bioactive film material and a medical double-sided adhesive tape that are attached together, wherein the medical double-sided adhesive tape comprises the following components and dosages: acrylic acid 30%, collagen specific binding peptide 2%-5%, elastin specific binding peptide 2%-5%, isocyanatoethyl methacrylate 1%-5%, N-acryloyloxy succinimide 1%-5%, gelatin methacrylate 1%-5%, hydroxypropyl methylcellulose 0.5%-5%, alpha-ketoglutaric acid 0.5%-5%, and magnesium ion compound 0.1%-0.5%, and the rest is deionized water. In the application, the composite bioactive film material reduces the water absorption expansion rate of the medical double-sided adhesive tape, improves the adhesion to the bioactive film material, and meets the requirement of long-term adhesion effect in the local environment rich in water on the human body wound.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medical materials, and particularly relates to a composite bioactive membrane material and a preparation method and application thereof. BACKGROUND

[0002] Amniotic membrane is the innermost layer of placenta, containing epithelial cells, smooth, no blood vessels, nerves and lymph, having certain elasticity, and being about 0.02-0.5mm thick. Its components mainly include collagen, fibronectin, laminin, elastin and mucopolysaccharide, etc. Amniotic membrane has wide application in regenerative medicine, for example, can be used as a natural scaffold conducive to cell growth adhesion, or as a source of various types of stem cells and growth factors.

[0003] Amniotic membrane has broad clinical application in wound care, including wound management and postoperative repair in ophthalmology, orthopedics, dermatology and stomatology. Among them, amniotic membrane in animal experiments and clinical observation related to ophthalmic diseases shows that amniotic membrane can promote corneal epithelial cell migration, inhibit neovascular proliferation, and has anti-inflammatory function. Fresh amniotic membrane contains growth factors, which can promote epithelialization, be conducive to the differentiation, migration of epithelial cells and enhance the adhesion of epithelial cells. Amniotic membrane can prevent leukocyte infiltration, inhibit the activity of various proteases such as trypsin, fibrinolysin, collagenase, etc., thereby reducing the degree of inflammation, shortening the duration of inflammation and inhibiting the formation of neovascularization. In summary, amniotic membrane can be used as a reliable donor material in ophthalmic clinical application. The existing amniotic membrane medical device product approved by China Food and Drug Administration is in the form of a membrane sheet, which is suitable for diseases in the field of ophthalmology, including pterygium, chemical injury, thermal burn, keratitis, corneal ulcer, symblepharon, glaucoma, bullous keratopathy, etc.

[0004] However, the existing amniotic membrane product needs surgical suture, which on the one hand increases the difficulty of surgical operation for doctors, and on the other hand causes secondary trauma to patients due to suture operation. In order to promote the application of amniotic membrane product and avoid the trauma to patients caused by suture type amniotic membrane graft, suture-free type amniotic membrane material is gradually reported in the literature.

[0005] The paper "Sutureless contact lens-type amniotic membrane for persistent epithelial defects after infectious keratitis" (International journal of ophthalmology, 2022, 15(8), 1404-1406) reports a treatment method of sutureless contact lens-type amniotic membrane for persistent epithelial defects after infectious keratitis. The sutureless contact lens-type amniotic membrane is composed of a membrane-shaped amniotic membrane and a soft contact lens. It is obtained by physical molding and non-chemical cross-linking to achieve the basic curvature and requirements of a soft contact lens. The results show that the sutureless contact lens-type amniotic membrane can treat persistent epithelial defects after infectious keratitis without surgery. However, the amniotic membrane in this product is only physically attached to the soft contact lens substrate, which may not be firmly attached.

[0006] Combining membrane-shaped amniotic membrane with medical glue is another direction for suture-free amniotic membrane development. The paper "Dry double-sided tape for adhesion of wet tissues and devices" (Nature, 2019, 575(7781), 169-174.) and Chinese patent "Dry double-sided tape for adhesion of wet tissues and devices" (Application number: 202080049580.7) disclose a method for preparing a medical double-sided tape for bonding wet tissues and devices. The steps include: dissolving 30% (w / w) acrylic acid, 10% (w / w) gelatin, 1% (w / w) N-acryloyloxy succinimide, 0.1% (w / w) gelatin methacrylate and 0.2% (w / w) alpha-ketoglutaric acid in deionized water. Then filter the mixture with a 0.4 µm sterile syringe filter and pour it into a glass mold with spacing. The medical double-sided tape is cured in a UV lamp chamber (284 nm, 10 W power) for 20 minutes and completely dried. Finally, the medical double-sided tape is sealed in a plastic bag with a desiccant (silica gel package). However, this medical double-sided tape has a high water absorption swelling rate (more than 1000%), which makes it unsuitable for use in the development of suture-free amniotic membranes. When used in a rich body fluid environment (such as the ocular surface), it will swell and result in poor adhesion.

[0007] The patent "Functional tissue engineering material for nerve repair and preparation method thereof" (application number: 201310425493.9) discloses a functional tissue engineering material for nerve repair, which comprises human amniotic membrane as a carrier, nerve repair promoting neurotrophic factors specifically combined with collagen, and an inhibitor of calpain fixedly combined. The nerve repair promoting neurotrophic factors comprise one or more of brain-derived neurotrophic factor, nerve growth factor, neurotrophic factor 3 and glial cell-derived neurotrophic factor; the neurotrophic factor is a fusion protein of neurotrophic factor and collagen specific binding domain constructed by a genetic engineering method. The gene sequence of the collagen specific binding domain CBD is TKKTLRT. The technology only discloses the use of polypeptide with specific collagen binding domain and other functional factors for superposition recombination, and the polypeptide with specific collagen binding domain only has coordination action binding force with amniotic membrane collagen. The binding force is a weak interaction.

[0008] In summary, it is urgent to develop a composite bioactive membrane material to overcome the problems of high water absorption and swelling rate of the medical double-sided tape and poor adhesion to amniotic membrane and human tissues in the prior art, and to better meet the clinical needs. SUMMARY

[0009] To solve the above technical problems, the present application provides a composite bioactive membrane material and a preparation method and application thereof, which reduces the water absorption and swelling rate of the medical double-sided tape, improves the adhesion to the bioactive membrane material, meets the requirement of long-term adhesion effect in the local environment rich in water of the human body wound, and further promotes wound healing and postoperative repair. The water absorption and swelling rate index of the composite bioactive membrane material is less than 300%, the adhesion index after immersion in normal saline for one week is greater than 1.5N, the technical problem of poor adhesion is solved, and the preparation method is simple and low in cost.

[0010] The composite bioactive membrane material provided in the present application to solve the above technical problems has the following characteristics: the composite bioactive membrane material comprises a bioactive membrane material and a medical double-sided tape which are attached together, and the medical double-sided tape comprises the following components and amounts by mass concentration: acrylic acid 30%, collagen specific binding peptide 2%-5%, elastin specific binding peptide 2%-5%, isocyanatoethyl methacrylate 1%-5%, N-acryloyloxy succinimide 1%-5%, gelatin methacrylate 1%-5%, hydroxypropyl methylcellulose 0.5%-5%, alpha-ketoglutaric acid 0.5%-5%, and magnesium ion compound 0.1%-0.5%, and the rest is deionized water.

[0011] The bioactive membrane material comprises any one of amniotic membrane, small intestine mucosa, pericardial membrane, skin acellular dressing, chitosan and gelatin.

[0012] The amino acid sequence of the collagen-specific binding peptide is RRKEEDEKED, and the corresponding three-letter amino acid sequence is Arg-Arg-Lys-Glu-Glu-Asp-Glu-Lys-Glu-Asp, and the structural formula is as follows:

[0013] ,

[0014] The amino acid sequence of the elastin-specific binding peptide is HHKEDDRRHK, and the corresponding three-letter amino acid sequence is His-His-Lys-Glu-Asp-Asp-Arg-Arg-His-Lys, and the structural formula is as follows:

[0015] ,

[0016] The hydroxypropyl methyl cellulose is a hydrophilic macromolecule with an average molecular weight of 90KDa-120KDa;

[0017] The magnesium ion compound is magnesium chloride or magnesium sulfate.

[0018] The acrylic acid is a hydrophilic polymerizable monomer, the isocyanatoethyl methacrylate is a hydrophobic polymerizable monomer, and the amine coupling agent is used.

[0019] The hydroxypropyl methyl cellulose aqueous solution has low temperature sensitivity, and is used in the application to improve the uniformity of the prepared medical double-sided adhesive tape finished product.

[0020] The collagen-specific binding peptide and the elastin-specific binding peptide realize the combination of collagen and elastin in the human wound local tissue and the bioactive membrane material, and the combination is realized by the receptor-ligand spatial coordination and electrostatic combination between proteins.

[0021] The magnesium ion compound provides the ion bond combination between the collagen-specific binding peptide, the elastin-specific binding peptide, the acrylic acid hydrophilic polymer, the bioactive membrane material and the human wound local tissue, as shown in the schematic Figure 5 .

[0022] In the application, the N-acryloyloxy succinimide is an amine coupling agent, the gelatin methacrylate is a crosslinking agent, and the alpha-ketoglutaric acid is a photoinitiator.

[0023] The preparation method of the medical double-sided tape described in this invention is as follows: acrylic acid, collagen-specific binding peptide, elastin-specific binding peptide, isocyanate methacrylate, N-acryloyloxysuccinimide, gelatin methacrylate, hydroxypropyl methylcellulose, α-ketoglutaric acid, and magnesium ion compound are dissolved in deionized water and mixed evenly. The mixture is then filtered through a 0.4µm sterile syringe filter and poured into a spaced glass mold. The medical double-sided tape is cured in an ultraviolet lamp chamber at 365 nm, 85W power, and 20°C~60°C for 55-65 minutes, and then dried in an oven at 45-55°C for 15-25 minutes.

[0024] The present invention discloses a method for preparing a composite bioactive membrane material, characterized by comprising the following steps:

[0025] (1) Prepare bioactive membrane materials;

[0026] (2) Preparation of medical double-sided adhesive tape;

[0027] (3) Apply medical double-sided tape to the outer periphery of the bioactive membrane material, with a coverage area of ​​1 / 16 to 1 / 4 of the bioactive membrane material.

[0028] The present invention relates to the application of a composite bioactive membrane material in the preparation of materials for wound management and postoperative repair in ophthalmology, orthopedics, dermatology and dentistry.

[0029] The application of a composite bioactive membrane material in this invention can also be applied to sutureless amniotic membrane products for the treatment of ocular surface diseases. In this invention, medical double-sided tape is used to bond the amniotic membrane and the bandage lens to prevent the amniotic membrane from slipping off the ocular surface without sutures.

[0030] The beneficial effects of the adhesive, stitchless composite bioactive membrane material of this invention are as follows:

[0031] (1) Compared with existing medical double-sided tape technology (paper: Nature, 2019, 575(7781), 169–174; patent #: 202080049580.7), the water absorption swelling rate is significantly reduced (less than 300%), which meets the requirement of maintaining the adhesive effect for a long time (more than one week) in a water-rich environment in the body. The principle of reducing water absorption swelling is that the addition of isocyanate ethyl methacrylate hydrophobic monomer reduces the water absorption swelling percentage of medical double-sided tape; the addition of collagen-specific binding peptide, elastin-specific binding peptide, and magnesium ion compound increases the binding points inside the tape, thereby reducing water penetration.

[0032] (2) In the adhesion principle of the medical double-sided tape, the medical double-sided tape technology disclosed by the application comprises the following adhesion principles: hydrogen bonds and electrostatic interactions provided by the acrylic hydrophilic polymer and the hydroxypropyl methyl cellulose, covalent bonds generated by the combination of N-acryloyloxy succinimide polymer and isocyanate methacrylate polymer with amino groups in biological tissues, receptor-ligand spatial coordination and electrostatic bonding between specific collagen-binding peptides and elastin-specific binding peptides and corresponding proteins, and ionic bonding between collagen-specific binding peptides, elastin-specific binding peptides, acrylic hydrophilic polymers, bioactive films and local tissues of human wounds provided by magnesium ion compounds. The above four adhesion principles effectively improve the adhesion to biological tissues, and the adhesion force remains greater than 1.5N after immersion in physiological saline for one week.

[0033] (3) The uniformity of the prepared medical double-sided tape product is improved. In the comparative technology (paper: Nature, 2019, 575(7781), 169-174; patent #: 202080049580.7), the gelatin aqueous solution is very sensitive to temperature, for example, a gelatin pre-prepared aqueous solution with a mass concentration of 10% will solidify and settle at room temperature (25 degrees Celsius), which is difficult to ensure the uniformity of the prepared medical double-sided tape product. The hydroxypropyl methyl cellulose aqueous solution in the application has low temperature sensitivity, which is conducive to improving the uniformity of the prepared medical double-sided tape product.

[0034] The adhesive composite bioactive film in the application has wide clinical application in wound care, including wound management and postoperative repair in ophthalmology, orthopedics, dermatology and oral surgery. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 is a water absorption expansion rate test result graph of the medical double-sided tape;

[0036] Figure 2 is a schematic diagram of the adhesion force test method of the medical double-sided tape in the application;

[0037] Figure 3 is a graph of the adhesion force test results of the medical double-sided tape;

[0038] Figure 4 is a graph of the long-term adhesion performance test results of the medical double-sided tape in the application;

[0039] (Note: The left graph shows that the medical double-sided tape used in Comparative Example 1 is used to adhere two pieces of pigskin, and after immersion in physiological saline for one week, the medical double-sided tape is severely water-absorbed and expanded, and the adhesion is not firm, resulting in separation; the right graph shows that the medical double-sided tape used in the preferred embodiment 2 in the application is used to adhere two pieces of pigskin, and after immersion in physiological saline for one week, the adhesion can still be maintained)

[0040] Figure 5 The principle diagram of the magnesium ion compound in the present application. DETAILED DESCRIPTION

[0041] The present application is further described below in conjunction with specific embodiments, wherein the membrane-shaped amniotic membrane is purchased from the market. It should be noted that the "bioactive membrane material" in the listed examples of the present application only lists "bioamniotic membrane" as an example. As mentioned earlier, the "bioactive membrane material" includes but is not limited to amniotic membrane, small intestine mucosa, pericardial membrane, skin acellular dressing, chitosan, gelatin, and the "medical double-sided tape" described in the present application has adhesive properties for various membrane materials and does not limit the type of membrane material.

[0042] Example 1:

[0043] The steps include:

[0044] (1) Preparation of medical double-sided tape;

[0045] Dissolve 30% acrylic acid (all concentrations are mass percent concentrations), 2% collagen-specific binding peptide (amino acid sequence RRKEEDEKED, purchased from Shanghai Qiangyao Biological Technology Co., Ltd.), 2% elastin-specific binding peptide (amino acid sequence HHKEDDRRHK, purchased from Shanghai Qiangyao Biological Technology Co., Ltd.), 1% isocyanatoethyl methacrylate, 1% N-acryloyl succinimide, 1% gelatin methacrylate, 0.5% hydroxypropyl methylcellulose, 0.5% alpha-ketoglutaric acid, and 0.1% magnesium chloride in deionized water. Then filter the mixture with a 0.4 µm sterile syringe filter and pour it into a glass mold with spacing. The medical double-sided tape is cured in a UV lamp chamber (365 nm, 85 W power) at 20°C for 60 minutes and dried in a 50°C oven for 20 minutes. Finally, the medical double-sided tape is sealed in a plastic bag with a desiccant (silica gel package).

[0046] (2) Partially adhere the medical double-sided tape to one side of the membrane-shaped amniotic membrane.

[0047] The amniotic membrane is from a human. The membrane-shaped amniotic membrane is 1 cm x 1 cm square in size. First, immerse the membrane-shaped amniotic membrane in sterile normal saline for more than 5 minutes to maintain the wet state of the membrane-shaped amniotic membrane, then take out the amniotic membrane and partially adhere the medical double-sided tape to the outer periphery of the membrane-shaped amniotic membrane, with a coverage area of 1 / 16 of the membrane-shaped amniotic membrane.

[0048] Example 2:

[0049] The steps include:

[0050] (1) Preparation of medical double-sided tape;

[0051] Acrylic acid 30% (all concentrations are mass percent concentrations), collagen specific binding peptide (amino acid sequence RRKEEDEKED, purchased from Shanghai Qiangyao Biotechnology Co., Ltd.) 5%, elastin specific binding peptide (amino acid sequence HHKEDDRRHK, purchased from Shanghai Qiangyao Biotechnology Co., Ltd.) 5%, isocyanatoethyl methacrylate 5%, N-acryloyloxysuccinimide 5%, gelatin methacrylate 5%, hydroxypropyl methylcellulose 5%, alpha-ketoglutaric acid 5%, and magnesium chloride 0.5% were dissolved in deionized water. Then the mixture was filtered with a 0.4 pm sterile syringe filter and poured into a glass mold with spacing. The medical double-sided tape was cured in an ultraviolet lamp chamber (365 nm, 85 W power) at 20 °C for 60 minutes and dried in a 50 °C oven for 20 minutes. The final medical double-sided tape was sealed in a plastic bag with a desiccant (silica gel packet).

[0052] (2) The medical double-sided tape was partially adhered to one side of the membrane-shaped amniotic membrane.

[0053] The amniotic membrane was obtained from a human. The membrane-shaped amniotic membrane had a size of 3 cm x 3 cm square. First, the membrane-shaped amniotic membrane was soaked in sterile normal saline for more than 5 minutes to keep the membrane-shaped amniotic membrane in a wet state, and then the amniotic membrane was taken out and the medical double-sided tape was partially adhered to the outer periphery of the membrane-shaped amniotic membrane, covering an area of 1 / 8 of the membrane-shaped amniotic membrane.

[0054] Example 3:

[0055] including the following steps:

[0056] (1) Preparation of medical double-sided tape;

[0057] Acrylic acid 30% (all concentrations are mass percent concentrations), collagen specific binding peptide (amino acid sequence RRKEEDEKED, purchased from Shanghai Qiangyao Biotechnology Co., Ltd.) 3%, elastin specific binding peptide (amino acid sequence HHKEDDRRHK, purchased from Shanghai Qiangyao Biotechnology Co., Ltd.) 3%, isocyanatoethyl methacrylate 3%, N-acryloyloxysuccinimide 2%, gelatin methacrylate 2%, hydroxypropyl methylcellulose 2%, alpha-ketoglutaric acid 3%, and magnesium chloride 0.3% were dissolved in deionized water. Then the mixture was filtered with a 0.4 pm sterile syringe filter and poured into a glass mold with spacing. The medical double-sided tape was cured in an ultraviolet lamp chamber (365 nm, 85 W power) at 60 °C for 60 minutes and dried in a 50 °C oven for 20 minutes. The final medical double-sided tape was sealed in a plastic bag with a desiccant (silica gel packet).

[0058] (2) The medical double-sided tape was partially adhered to one side of the membrane-shaped amniotic membrane.

[0059] Amnion was from human. The size of the membrane amnion was 3 cm x 3 cm square. Partial adhesion was that the membrane amnion was first soaked in sterile saline for more than 5 minutes to keep the membrane amnion in a wet state, and then the amnion was taken out to partially adhere the medical double-sided tape to the periphery of the membrane amnion, with a coverage area of 1 / 4 of the membrane amnion.

[0060] Example 4:

[0061] comprising the following steps:

[0062] (1) Preparation of medical double-sided tape;

[0063] Acrylic acid 30% (all concentrations are mass percent concentrations), collagen-specific binding peptide (amino acid sequence RRKEEDEKED, purchased from Shanghai Qiangyao Biotechnology Co., Ltd.) 5%, elastin-specific binding peptide (amino acid sequence HHKEDDRRHK, purchased from Shanghai Qiangyao Biotechnology Co., Ltd.) 5%, isocyanatoethyl methacrylate 5%, N-acryloyloxy succinimide 5%, gelatin methacrylate 5%, hydroxypropyl methylcellulose 5%, a-ketoglutaric acid 5%, and magnesium chloride 0.5% were dissolved in deionized water. Then the mixture was filtered with a 0.4 μm sterile syringe filter, and poured onto a glass mold with spacing. The medical double-sided tape was cured in an ultraviolet lamp chamber (365 nm, 85 W power) at 50°C for 60 minutes, and dried in a 50°C oven for 20 minutes. Finally, the medical double-sided tape was sealed in a plastic bag with a desiccant (silica gel package).

[0064] (2) Partially adhere the medical double-sided tape to one side of the membrane amnion.

[0065] Amnion was from pig. The size of the membrane amnion was a circle with a diameter of 3 cm. Partial adhesion was that the membrane amnion was first soaked in sterile saline for more than 5 minutes to keep the membrane amnion in a wet state, and then the amnion was taken out to partially adhere the medical double-sided tape to the periphery of the membrane amnion, with a coverage area of 1 / 8 of the membrane amnion.

[0066] Example 5:

[0067] comprising the following steps:

[0068] (1) Preparation of medical double-sided tape;

[0069] Acrylic acid 30%, collagen specific binding peptide (amino acid sequence: RRKEEDEKED, purchased from Shanghai Qiangyao Biotechnology Co., Ltd.) 5%, elastin specific binding peptide (amino acid sequence: HHKEDDRRHK, purchased from Shanghai Qiangyao Biotechnology Co., Ltd.) 5%, isocyanatoethyl methacrylate 5%, N-acryloyloxysuccinimide 5%, gelatin methacrylate 5%, hydroxypropyl methylcellulose 5%, alpha-ketoglutaric acid 5%, and magnesium chloride 0.5% were dissolved in deionized water. Then the mixture was filtered with a 0.4 μm sterile syringe filter, and poured into a glass mold with spacing. The medical double-sided tape was cured under a UV lamp chamber (365 nm, 85W power) at 50°C for 60 minutes, and dried in a 50°C oven for 20 minutes. Finally, the medical double-sided tape was sealed in a plastic bag with desiccant (silica gel package).

[0070] (2) Partially adhere the medical double-sided tape to one side of the amniotic membrane.

[0071] The amniotic membrane was obtained from a cow. The amniotic membrane was in the form of a circular sheet with a diameter of 1 cm. Partial adhesion was achieved by first soaking the amniotic membrane in sterile normal saline for more than 5 minutes, keeping the amniotic membrane moist, then removing the amniotic membrane and partially adhering the medical double-sided tape to the periphery of the amniotic membrane, covering an area of 1 / 8 of the amniotic membrane.

[0072] Example 6:

[0073] A method for preparing a composite bioactive membrane material is as described in Example 1, wherein the medical double-sided tape is composed of the following components and amounts: acrylic acid 30%, collagen specific binding peptide 2.2%, elastin specific binding peptide 3.5%, isocyanatoethyl methacrylate 2.5%, N-acryloyloxysuccinimide 2.6%, gelatin methacrylate 2.5%, hydroxypropyl methylcellulose 3.5%, alpha-ketoglutaric acid 4.5%, and magnesium chloride 0.3%, with the rest being deionized water. The medical double-sided tape was cured under a UV lamp chamber (365 nm, 85W power) at 45°C for 55 minutes, and dried in a 45°C oven for 25 minutes.

[0074] Comparative Example 1:

[0075] Comparative Example 1 is a medical double-sided tape prepared according to the method described in the reference paper (Nature, 2019, 575(7781), 169-174) and the patent (Application No. 202080049580.7).

[0076] The method comprises the following steps:

[0077] (1) Preparing a medical double-sided tape;

[0078] Acrylic acid 30% (w / w), gelatin 10% (w / w), N-acryloyloxysuccinimide 1% (w / w), gelatin methacrylate 0.1% (w / w) and a-ketoglutaric acid 0.2% (w / w) were dissolved in deionized water. Then the mixture was filtered with 0.4 pm sterile syringe filter and poured into a glass mold with spacing. The medical adhesive tape was cured in a UV lamp chamber (284 nm, 10 W power) for 20 minutes and completely dried. The final medical adhesive tape was sealed in a plastic bag with desiccant (silica gel packet).

[0079] (2) The medical adhesive tape was partially adhered to one side of the amniotic membrane.

[0080] The amniotic membrane was from human. The size of the amniotic membrane was 3 cm x 3 cm square. Partial adhesion was first soaking the amniotic membrane in sterile normal saline for more than 5 minutes, keeping the amniotic membrane in a wet state, then taking out the amniotic membrane and partially adhering the medical adhesive tape to the periphery of the amniotic membrane, with a coverage area of 1 / 8 of the amniotic membrane.

[0081] Comparative Example 2:

[0082] The medical adhesive tape was prepared according to the collagen-specific knot and peptide preparation in the reference patent “Functional tissue engineering material for nerve repair and its preparation method” (Application No. 201310425493.9).

[0083] The following steps were included:

[0084] (1) Preparation of medical adhesive tape;

[0085] Acrylic acid 30% (all concentrations are mass percent), collagen-specific binding peptide (amino acid sequence TKKTLRT, purchased from Shanghai Qiangyao Biological Technology Co., Ltd.) 5%, isocyanatoethyl methacrylate 5%, N-acryloyloxysuccinimide 5%, gelatin methacrylate 5%, hydroxypropyl methyl cellulose 5%, a-ketoglutaric acid 5% and magnesium chloride 0.5% were dissolved in deionized water. Then the mixture was filtered with 0.4 pm sterile syringe filter and poured into a glass mold with spacing. The medical adhesive tape was cured in a UV lamp chamber (365 nm, 85 W power) and 60°C for 60 minutes, and dried in a 50°C oven for 20 minutes. The final medical adhesive tape was sealed in a plastic bag with desiccant (silica gel packet).

[0086] (2) The medical adhesive tape was partially adhered to one side of the amniotic membrane.

[0087] The amniotic membrane is derived from human. The size of the membrane patch is 3 cm x 3 cm square. The partial adhesion is to first soak the membrane patch in sterile saline for more than 5 minutes, keep the membrane patch in a wet state, then take out the amniotic membrane and partially adhere the medical double-sided tape to the periphery of the membrane patch, with a coverage area of 1 / 8 of the membrane patch.

[0088] Comparative Example 3:

[0089] Comparative Example 3 is a medical double-sided tape prepared with isocyanatoethyl methacrylate hydrophobic monomer, but without specific binding peptide and magnesium ion compound.

[0090] The steps include:

[0091] (1) Preparation of medical double-sided tape;

[0092] Acrylic acid 30% (all concentrations are mass percent), isocyanatoethyl methacrylate 5%, N-acryloyloxy succinimide 5%, gelatin methacrylate 5%, hydroxypropyl methylcellulose 5%, and a-ketoglutaric acid 5% were dissolved in deionized water. Then the mixture was filtered with a 0.4 µm sterile syringe filter, and poured into a glass mold with spacing. The medical double-sided tape was cured in a UV lamp chamber (365 nm, 85 W power) at 60°C for 60 minutes, and dried in a 50°C oven for 20 minutes. Finally, the medical double-sided tape was sealed in a plastic bag with a desiccant (silica gel package).

[0093] (2) Partially adhere the medical double-sided tape to one side of the membrane patch.

[0094] The amniotic membrane is derived from human. The size of the membrane patch is 3 cm x 3 cm square. The partial adhesion is to first soak the membrane patch in sterile saline for more than 5 minutes, keep the membrane patch in a wet state, then take out the amniotic membrane and partially adhere the medical double-sided tape to the periphery of the membrane patch, with a coverage area of 1 / 8 of the membrane patch.

[0095] Comparative Example 4:

[0096] Comparative Example 4 is a medical double-sided tape prepared with specific binding peptide and magnesium ion compound, but without isocyanatoethyl methacrylate hydrophobic monomer.

[0097] The steps include:

[0098] (1) Preparation of medical double-sided tape;

[0099] Acrylic acid 30% (all concentrations are mass percent concentrations), collagen specific binding peptide (amino acid sequence RRKEEDEKED, purchased from Shanghai Qiangyao Biotechnology Co., Ltd.) 5%, elastin specific binding peptide (amino acid sequence HHKEDDRRHK, purchased from Shanghai Qiangyao Biotechnology Co., Ltd.) 5%, N-acryloyloxysuccinimide 5%, gelatin methacrylate 5%, hydroxypropyl methylcellulose 5%, alpha-ketoglutaric acid 5%, and magnesium chloride 0.5% were dissolved in deionized water. Then the mixture was filtered with a 0.4 µm sterile syringe filter, and poured into a glass mold with spacing. The medical double-sided tape was cured under a UV lamp chamber (365 nm, 85W power) at 60°C for 60 minutes, and dried in a 50°C oven for 20 minutes. The final medical double-sided tape was sealed in a plastic bag with desiccant (silica gel packet).

[0100] (2) Partially adhere the medical double-sided tape to one side of the amniotic membrane.

[0101] The amniotic membrane was from human. The amniotic membrane was in the form of a membrane sheet with a size of 3 cm x 3 cm square. The partial adhesion was first to soak the membrane sheet in sterile normal saline for more than 5 minutes, keeping the membrane sheet in a wet state, then taking out the amniotic membrane and partially adhering the medical double-sided tape to the periphery of the membrane sheet, with a coverage area of 1 / 8 of the membrane sheet.

[0102] Comparative Example 5:

[0103] Comparative Example 5 was a medical double-sided tape containing only the collagen specific binding peptide described in the present application, but not containing the elastin specific binding peptide.

[0104] Including the following steps:

[0105] (1) Prepare the medical double-sided tape;

[0106] Acrylic acid 30% (all concentrations are mass percent concentrations), collagen specific binding peptide (amino acid sequence RRKEEDEKED, purchased from Shanghai Qiangyao Biotechnology Co., Ltd.) 5%, isocyanatoethyl methacrylate 5%, N-acryloyloxysuccinimide 5%, gelatin methacrylate 5%, hydroxypropyl methylcellulose 5%, alpha-ketoglutaric acid 5%, and magnesium chloride 0.5% were dissolved in deionized water. Then the mixture was filtered with a 0.4 µm sterile syringe filter, and poured into a glass mold with spacing. The medical double-sided tape was cured under a UV lamp chamber (365 nm, 85W power) at 60°C for 60 minutes, and dried in a 50°C oven for 20 minutes. The final medical double-sided tape was sealed in a plastic bag with desiccant (silica gel packet).

[0107] (2) Partially adhere the medical double-sided tape to one side of the amniotic membrane.

[0108] The amniotic membrane is from human. The size of the membrane is 3cm x 3cm square. First, the membrane is soaked in sterile saline for more than 5 minutes to keep the membrane wet, and then the membrane is taken out and the medical double-sided tape is partially adhered to the outer periphery of the membrane, covering an area of 1 / 8 of the membrane.

[0109] Comparative Example 6:

[0110] Comparative Example 6 is a medical double-sided tape consistent with the components and process of Example 2, but the medical double-sided tape is completely adhered to one side of the membrane, i.e. the medical double-sided tape covers 100% of one side of the membrane.

[0111] The technical performance indicators of the medical double-sided tape described in the present application, and the comparison with the existing medical double-sided tape technology are shown in the following table:

[0112] Table 1 Technical Comparison Table

[0113] Existing medical double-sided tape technology 1 Characteristics and limitations Technical features and improvements of the medical double-sided tape Components in typical embodiments Acrylic acid, gelatin, N-acryloyloxysuccinimide, gelatin methacrylate, alpha-ketoglutaric acid Acrylic acid, collagen-specific binding peptide, elastin-specific binding peptide, isocyanatoethyl methacrylate, N-acryloyloxysuccinimide, gelatin methacrylate, hydroxypropyl methylcellulose, alpha-ketoglutaric acid, and magnesium ion compound Water absorption and swelling rate index 2 ]] More than 1000% Less than 300% Principle of reducing water absorption and swelling --- (1) The addition of isocyanatoethyl methacrylate hydrophobic monomer reduces the water absorption and swelling percentage of the medical double-sided tape; (2) The addition of collagen-specific binding peptide, elastin-specific binding peptide, and magnesium ion compound improves the adhesion of the tape, thereby reducing water infiltration. Adhesion principle (1) Hydrogen bonds and electrostatic interactions provided by acrylic acid hydrophilic polymer and gelatin; (2) Covalent bonds produced by N-acryloyloxysuccinimide polymer binding with amino groups in biological tissues. (1) Hydrogen bonds and electrostatic interactions provided by acrylic acid hydrophilic polymer and hydroxypropyl methylcellulose; (2) Covalent bonds produced by N-acryloyloxysuccinimide polymer and isocyanatoethyl methacrylate polymer binding with amino groups in biological tissues; (3) Receptor-ligand spatial coordination and electrostatic bonding between collagen-specific binding peptide and elastin-specific binding peptide; (4) Ionic bonding between collagen-specific binding peptide, elastin-specific binding peptide, acrylic acid hydrophilic polymer, amniotic membrane, and local tissues on the human wound surface provided by magnesium ion compound. Adhesion force index after one day immersion in physiological saline 3 ]] Less than 0.05 N More than 1.5 N Preparation solution stability Gelatin aqueous solution is very sensitive to temperature, for example, a 10% mass concentration gelatin aqueous solution will solidify and settle at room temperature (25 degrees Celsius), making it difficult to ensure the uniformity of the prepared medical double-sided tape product Hydroxypropyl methylcellulose aqueous solution has lower temperature sensitivity, which is beneficial to improving the uniformity of the prepared medical double-sided tape product.

[0114] (Note: "Existing medical double-sided tape technology" refers to the relevant technology disclosed in the paper: Nature, 2019, 575(7781), 169-174 and patent # 202080049580.7; "Water absorption and swelling rate index" is the data obtained by using the method in "Test Example 1 Water absorption and swelling rate test of medical double-sided tape" described in the present application; "Adhesion force index after immersion in saline for one day" is the data obtained by using the method in "Test Example 2 Adhesion force test of medical double-sided tape" described in the present application)

[0115] Test Example 1 Water absorption and swelling rate test of medical double-sided tape

[0116] The test includes the following materials: Examples 1-5 and Comparative Examples 1-5. Among them, Examples 1-5 are medical double-sided tapes prepared by the method described in the present application. Comparative Example 1 is a medical double-sided tape prepared by the method in the reference paper (Nature, 2019, 575(7781), 169-174) and patent (Application No. 202080049580.7). Comparative Example 2 is a medical double-sided tape prepared by collagen-specific knots and peptides in the reference patent “Functional tissue engineering material for nerve repair and its preparation method” (Application No. 201310425493.9). Comparative Example 3 is a medical double-sided tape containing isocyanatoethyl methacrylate hydrophobic monomers, but does not contain specific binding peptides and magnesium ion compounds. Comparative Example 4 is a medical double-sided tape containing specific binding peptides and magnesium ion compounds, but does not contain isocyanatoethyl methacrylate hydrophobic monomers. Comparative Example 5 is a medical double-sided tape containing only the collagen-specific binding peptides described in the present application, but does not contain elastin-specific binding peptides.

[0117] Test method:

[0118] After the medical double-sided tapes were dried in the oven, they were weighed and then soaked in physiological saline at 37°C for one week. After being taken out, the surface was wiped with filter paper to remove excess liquid, and then re-weighed. The water absorption swelling rate was calculated using the following formula:

[0119] ,

[0120] Ww and Wd represent the mass of the wet sample and dry sample after soaking for one week, respectively.

[0121] The experimental results are shown in Table 2 and Figure 1 .

[0122] Table 2

[0123]

[0124] The experimental results show that the water absorption swelling rate of the medical double-sided tapes in Examples 1-5 is greatly reduced (from more than 900% to less than 300%) compared to the medical double-sided tapes in Comparative Examples 1-5. Comparative Example 2 is a medical double-sided tape prepared by collagen-specific knots and peptides in the reference patent “Functional tissue engineering material for nerve repair and its preparation method” (Application No. 201310425493.9). Since its technical principle only considers the coordination action of collagen-specific knots and peptides with human amniotic collagen binding force, this binding force has very little effect on inhibiting water absorption swelling (the water absorption swelling rate is still as high as 1022%).

[0125] The effect of reducing water swelling rate by relying on a single technique of reducing water swelling rate in Comparative Example 3 (addition of isocyanatoethyl methacrylate hydrophobic monomer to reduce the water swelling rate of the medical double-sided tape) or Comparative Example 4 (addition of collagen-specific binding peptide, elastin-specific binding peptide, and magnesium ion compound to increase the binding points inside the tape, thereby reducing water penetration) is very weak (from 1115% to 945% and 978%).

[0126] Compared with Comparative Example 5 (medical double-sided tape containing only the collagen-specific binding peptide described in the present application, but not containing the elastin-specific binding peptide), the water swelling rate of the medical double-sided tape in Examples 1-5 is greatly reduced (from 912% to below 300%).

[0127] The sequence design of the collagen-specific binding peptide and the elastin-specific binding peptide in the medical double-sided tape system of the present application further enhances the ionic bonding between the magnesium ion compound and improves the chemical cross-linking points in the internal network structure of the tape, achieving a significant reduction in water swelling rate; and in the present application, the corresponding specific binding peptides are separately optimized and designed for the two main protein components in amniotic membrane and human eye surface tissues, i.e., collagen and elastin, further enhancing the coordination force. The superposition of the two technical principles in the present application produces an unexpected technical effect of greatly reducing the water swelling rate (below 300%).

[0128] Test Example 2: Adhesion test of medical double-sided tape

[0129] This test includes the following materials: Examples 1-5 and Comparative Examples 1-5. Among them, Examples 1-5 are medical double-sided tapes prepared by the method described in the present application. Comparative Example 1 is a medical double-sided tape prepared by the method in the reference paper (Nature, 2019, 575(7781), 169-174) and patent (Application No.: 202080049580.7). Comparative Example 2 is a medical double-sided tape prepared by the collagen-specific binding peptide in the reference patent "Functional tissue engineering material for nerve repair and its preparation method" (Application No.: 201310425493.9). Comparative Example 3 is a medical double-sided tape containing isocyanatoethyl methacrylate hydrophobic monomer, but not containing specific binding peptide and magnesium ion compound. Comparative Example 4 is a medical double-sided tape containing specific binding peptide and magnesium ion compound, but not containing isocyanatoethyl methacrylate hydrophobic monomer. Comparative Example 5 contains only the collagen-specific binding peptide described in the present application, but not the elastin-specific binding peptide.

[0130] Test method: cut fresh pigskin into 1cm x 3cm strips, cut medical adhesive tape into 1cm x 1cm strips. After wetting the surface of the pigskin with normal saline, press the 1cm x 1cm medical adhesive tape to one end of the smooth surface of the two pieces of pigskin for 1 minute, immerse in normal saline for 1 day, and then take out. The other end of the two pieces of pigskin is clamped by the grips of a tensile testing machine. Record the maximum tensile force as the adhesion of the medical adhesive tape.

[0131] Figure 2 The adhesion test method of the medical adhesive tape in the present application is shown in the schematic diagram.

[0132] The experimental results are shown in Table 3 and Figure 3

[0133] Table 3

[0134]

[0135] The experimental results show that the medical adhesive tapes in Examples 1-5 effectively improve the adhesion compared to the medical adhesive tapes prepared by the methods in Comparative Examples 1-5, and can meet the requirements of maintaining adhesion effect in the water-rich environment of the ocular surface.

[0136] As can be seen from Table 3 above, if the effect of a single component in Comparative Example 2 (collagen-specific knot and peptide), Comparative Example 3 (isocyanatoethyl methacrylate hydrophobic monomer), and Comparative Example 4 (specific binding peptide and magnesium ion compound) is compared, the effect of improving adhesion in a water-rich environment in the present application is very significant, and the requirement that the adhesion can be effectively maintained after immersion in normal saline for one day is achieved.

[0137] Compared to Comparative Example 5 (medical adhesive tape containing only the collagen-specific binding peptide described in the present application, but not containing the elastin-specific binding peptide), the adhesion of the medical adhesive tapes in Examples 1-5 is greatly improved (from 0.32N to more than 1.5N).

[0138] ​From the overall technology, by the combination of two principles of reducing water absorption and swelling in the application (1) the addition of isocyanatoethyl methacrylate hydrophobic monomer reduces the water absorption and swelling percentage of the medical double-sided tape; (2) the addition of collagen-specific binding peptide, elastin-specific binding peptide and magnesium ion compound improves the binding points in the tape and thus reduces the penetration of water), and the combination of four adhesion principles, i.e. hydrogen bonds and electrostatic interactions provided by acrylic hydrophilic polymer and hydroxypropyl methyl cellulose; covalent bonds produced by the combination of N-acryloyloxy succinimide polymer and isocyanatoethyl methacrylate polymer with amino groups in biological tissues; receptor-ligand spatial coordination and electrostatic bonding between corresponding proteins provided by collagen-specific binding peptide and elastin-specific binding peptide; ionic bonding between collagen-specific binding peptide, elastin-specific binding peptide, acrylic hydrophilic polymer, amnion and local tissues of human wounds provided by magnesium ion compound, the requirements that the adhesion can be effectively maintained after immersion in physiological saline for 1 day are realized, and the effect on adhesion is very obvious.

[0139] Test Example 3: Long-term adhesion performance test of medical double-sided tape

[0140] This test includes the following materials: preferred example 2 and comparative example 1. Comparative example 1 is a medical double-sided tape prepared by the method in the reference paper (Nature, 2019, 575(7781), 169-174) and patent (application number: 202080049580.7).

[0141] Test method: cut fresh pigskin and medical double-sided tape into 1cm x 3cm strips. After wetting the surface of the pigskin with physiological saline, press the medical double-sided tape to the smooth surface of the two pieces of pigskin, then immerse the two pieces of pigskin in physiological saline at 37°C for 1 week. Then observe and take pictures, and record the adhesion state of the medical double-sided tape to the pigskin.

[0142] The experimental results are shown in Figure 4 Using the medical double-sided tape in comparative example 1 to adhere two pieces of pigskin immersed in physiological saline for 1 week resulted in serious water absorption and swelling of the medical double-sided tape, and the adhesion was not firm, resulting in separation. Using the medical double-sided tape in the preferred example 2 of the application to adhere two pieces of pigskin immersed in physiological saline for 1 week still maintained adhesion.

[0143] Test Example 4: Long-term adhesion performance test of local adhesion and overall adhesion

[0144] This test includes the following materials: preferred example 2 and comparative example 6. Comparative example 6 is a medical double-sided tape with the same components and process as example 2, but the medical double-sided tape is completely adhered to one side of the membrane-shaped amnion, i.e. the medical double-sided tape 100% covers one side of the membrane-shaped amnion.

[0145] Test method: the medical double-sided adhesive tape and the film-shaped amniotic membrane in example 2 and comparative example 6 were respectively immersed in normal saline in a 37℃ oven for 1 week, and then taken out to observe and record the adhesion state of both.

[0146] The experimental results show that: using the preferred example 2 in the application, the medical double-sided adhesive tape and the film-shaped amniotic membrane can still maintain adhesion after being immersed in water for 1 week, but the medical double-sided adhesive tape and the film-shaped amniotic membrane in comparative example 6 are separated after being immersed in water for 1 week. It shows that the partial adhesion method of the medical double-sided adhesive tape and the film-shaped amniotic membrane is superior to the complete adhesion method in maintaining adhesion force in underwater environment, because in the complete adhesion method, the medical double-sided adhesive tape will swell after absorbing water, which will cause extrusion with the film-shaped amniotic membrane, and then lead to the separation of the two, that is, the adhesion time is limited.

[0147] Test example 5: application of composite amniotic membrane material in ophthalmic surgery

[0148] Establishment of animal model: 20 New Zealand rabbits (2±0.5 kg) were anesthetized by intravenous injection of sodium pentobarbital, and the right eye was fixed by turning the body upward. The hair and eyelashes around the right eye were removed with a razor, and the area was disinfected with iodophor. The eye surface was locally anesthetized with obucaine, and the excess water on the cornea was absorbed with dry filter paper. A single layer of circular filter paper with a diameter of about 6 mm soaked in 1 mol / L H2SO4 solution was attached to the surface of the central region of the cornea, and the filter paper was removed after 30 seconds. After the excess acid solution on the cornea was absorbed with dry filter paper, the cornea was washed with a large amount of normal saline for 5 minutes. After one week of modeling, the models with mild burns that had self-healed and the models with full-thickness corneal burns that were too heavy were removed, and the models with mild burns were taken. The 16 models that could not be healed by self-repairing ability were used for repair experiments.

[0149] Surgical method: sterile surgical instruments were used for the operation, and the operation was strictly performed according to the requirements of clinical surgery.

[0150] (1) Adhesive suture-free composite amniotic membrane experimental group

[0151] Randomly take 8 burn models (2±0.5 kg) anesthetized by intravenous injection of sodium pentobarbital, and fix the front and hind limbs with a rope. The experimental rabbit was wrapped with a surgical drape, and the hair around the right eye of the rabbit was removed with an electric razor. The area was disinfected with iodophor to minimize the impact on the surgical process and postoperative corneal repair. The surgical site was the corneal acid burn model wound area. Under a microscope, the damaged site was first debrided, and the necrotic corneal epithelium and tissue were thoroughly cleaned with a diamond knife. The adhesive composite amniotic membrane prepared in the preferred example was laid flat and covered on the entire corneal surface, and pressed for 1 minute.

[0152] (2) Blank control group

[0153] Take the remaining 8 burn model, surgical method and experimental group the same surgical method, not using the sticky composite amniotic membrane material, the rest of the operation and postoperative care and the experimental group is the same.

[0154] Postoperative evaluation: observation and photography record the following evaluation items:

[0155] (1) every day to observe the mental state and activity of experimental rabbits, observation of corneal repair.

[0156] (2) animal experiment evaluation criteria: overall observation of corneal repair morphology, according to the following table classification of corneal repair in animal experiments, as shown in Table 4 below:

[0157] Table 4

[0158]

[0159] (Note: reference to the national eye injury professional eye disease group of the standard, and Roper-Hall on the degree of chemical injury, evaluation index for 0-1 level for effective, 2-4 level for invalid, overall effectiveness evaluation when invalid single item is invalid, evaluation of all data for statistical analysis)

[0160] (3) using the above "animal experiment evaluation criteria" evaluation method, 1 week, 4 weeks, 8 weeks after operation observation of blank control group and experimental group of corneal opacity and corneal neovascularization. 4 weeks and 8 weeks of sodium fluorescein staining in blank control group and experimental group, observation of corneal epithelial defect area. Comprehensive evaluation of the safety and effectiveness of the sticky composite amniotic membrane material repair ocular surface.

[0161] Results analysis:

[0162] (1) postoperative vascularization score results. As shown in Table 5 below:

[0163] Table 5

[0164]

[0165] (2) postoperative scarification score results, as shown in Table 6 below:

[0166] Table 6

[0167]

[0168] (3) postoperative epithelialization score results, as shown in Table 7 below:

[0169] Table 7

[0170]

[0171] The effectiveness comparison between the amniotic membrane group and the blank group is carried out by adopting a rabbit corneal acid burn model, and the results show that the adhesive composite amniotic membrane material experimental group can better assist in repairing the mild acid burn in the cornea.

[0172] Test Example 6: Application of the adhesive suture-free composite amniotic membrane material in skin defects

[0173] Twelve international standard experimental animal purebred healthy New Zealand white rabbits are selected, and the male and female are not limited, and the weight is 2.5-3.0 kg. Randomly divided into two treatment groups: the experimental group is implanted with the sample prepared in the preferred embodiment 2 of the application, and the control group is the Vaseline gauze group.

[0174] The specific experimental steps are as follows:

[0175] The back is depilated with 8% sodium sulfide solution, and after the success of intraperitoneal injection of 2% sodium pentobarbital (30 mg / kg) anesthesia, the skin of the surgical field on the back is disinfected, and the towel is laid. A 2x2 cm square full-thickness skin defect is made on both sides of the midline of the back, the left side is the experimental group, and the whole corneal surface is laid and pressed for 30 seconds. The right side is the control group, and the wound is covered with Vaseline gauze. After the operation, it is fixed.

[0176] Postoperative observation: observe the wound healing at 1W, 2W, 3W, 4W after the operation, measure the wound size, and calculate the wound healing rate. The healing area is taken at 4W after the operation and observed by pathology.

[0177] Wound healing rate = healing area / original wound area x 100

[0178] The results are as follows in Table 8:

[0179] Table 8: Comparison of wound healing rates between the experimental group and the control group

[0180]

[0181] From the above results, it can be seen that the adhesive composite amniotic membrane material provided by the application can accelerate the healing of the wound tissue compared with the traditional gauze.

[0182] Test Example 7: Application of the adhesive suture-free composite amniotic membrane material in tendon repair

[0183] Sixty international standard experimental animal purebred healthy New Zealand white rabbits are selected, and the male and female are not limited, and the weight is 2.5-3.0 kg. Randomly divided into three treatment groups: the experimental group is implanted with the sample prepared in the preferred embodiment 2 of the application, the control group 1 is the polylactic acid film group, and the control group 2 is the blank control group.

[0184] Specific experimental steps of the rabbit toe tendon animal model:

[0185] (1) Experimental grouping

[0186] Experimental group: 20 New Zealand white rabbits (2 ± 0.5 kg) were anesthetized with intravenous injection of sodium pentobarbital, and then placed in a fixation box with the left limb extended outside the box. The fur on the left hind limb of the rabbit was removed with a razor, and then disinfected with iodophor. The surgical site was the flexor tendon of the middle finger of the rabbit. A longitudinal incision was made at the surgical site with a surgical knife, and the flexor tendon was found. After being lifted with a vascular clamp, the tendon was sutured with a defect (about 1 / 2 of the width of the tendon) using Bunnell suture. The suture line used was 5-0 single polypropylene suture line. After suture, the sample prepared in Example 2 of the application was used to wrap the suture site. Finally, the surgical limb was fixed with plaster for 3 weeks.

[0187] Polylactic acid film group: 20 New Zealand white rabbits were operated on in the same way as the experimental group. After tendon suture, the suture site was wrapped with a polylactic acid anti-adhesion film, and then the peripheral wound was sutured and plastered.

[0188] Blank control group: 20 New Zealand white rabbits were operated on in the same way as the experimental group. After tendon suture, the peripheral wound was directly sutured and plastered.

[0189] All the above operations used sterile surgical instruments and were strictly in accordance with the requirements of clinical surgery. Within 3 days after the operation, the experimental animals were injected with cefazolin (40 mg / day) for antibiosis and carprophen (4 mg / kg) for pain relief. The mental state and activity of the experimental rabbits were observed every day.

[0190] (2) Postoperative sampling

[0191] At 3 weeks after the operation, the plasters of all the experimental rabbits were removed, and their free activities, especially the activities of the surgical limbs, were observed. The surgical toes were manually pulled, and the resistance to stretching and contraction of the toes was felt during the pulling process. Whether there was a significant difference between the normal toes and the surgical toes was observed.

[0192] At 3 weeks and 6 weeks after the operation, 10 rabbits were randomly selected from the experimental group, the polylactic acid film group and the blank control group for sampling observation. The surgical site was first incised with a surgical blade, and the tendon adhesion was observed. The animals were sacrificed by injecting 20 mL of air into the ear vein, and the surgical tendon was immediately removed. The tensile strength of the samples was tested using an electronic universal testing machine (model: RGM-6002T) at a speed of 5 mm / min. After the sample was destroyed, the computer automatically output the elastic modulus data of each group of specimens, and the biomechanical characteristics of the tendon were evaluated.

[0193] (3) Results of tendon biomechanical characteristics

[0194] The results of the tensile test analysis showed that the elastic modulus of the tendons of the experimental group, the polylactic acid film group and the blank control group was significantly improved at each observation time point. At 3 weeks after the operation, the elastic modulus (maximum breaking load) of the tendons of the experimental group was significantly better than that of the polylactic acid film group and the blank control group (P<0.05); at 6 weeks after the operation, there was no significant difference in the elastic modulus of the samples of the experimental group, the polylactic acid film group and the blank control group. It is shown that the amnion can effectively improve the tensile strength of the damaged tendon in the early repair stage, and the experimental group shows the best tendon repair effect. See Table 9 in detail:

[0195] Table 9 Results of the biomechanical characteristics of the tendons

[0196]

[0197] The experimental group was compared with the polylactic acid film group and the blank control group, respectively. .

[0198] Application of composite amnion material in spinal surgery

[0199] Sixty adult New Zealand rabbits with a body weight of 2.0-3.0 kg were selected without gender limitation. According to the different epidural coverings of the lamina defects, the 60 rabbits were randomly divided into an experimental group, a polylactic acid film group and a blank control group, with 20 rabbits in each group.

[0200] Under sterile operation, a posterior median incision with a length of about 20 mm was taken, and each layer was cut open and cut off. The L5 spinous process was cut off, and the L5 lamina was removed by using a micro-lamina bone rongeur to form a 10 mm*5 mm dura mater exposed area. The epidural fat was removed. After complete hemostasis, the dura mater was covered with the experimental group (the sample prepared in the preferred embodiment 2 in the application) and the polylactic acid film according to the preoperative random grouping, and the group without any interposition was used as the blank control group. All animals were fed in separate cages under the same postoperative conditions without taking any medicine.

[0201] Observation content and method:

[0202] Specimen collection and processing Five rabbits were sacrificed in each group at 2, 4, 8 and 12 weeks after the operation. After the animals were sacrificed, the L5 segment of the spinal column including the sacrospinal muscle and the vertebral body accessories was completely taken out, fixed in formalin solution and paraffin-embedded for sectioning with a thickness of 5 μm. The tissue sections were subjected to HE, Masson and picric acid-scarlet red staining, respectively.

[0203] Optical microscope observation The distribution of the epidural scar and the relationship with the dura mater and the nerve root were observed under an ordinary optical microscope.

[0204] Computer image analysis (Axiioplan2 image, USA) The cross-sectional area of the irregular scar was calculated, and the ratio of the scar area to the spinal canal area was determined as the scar index.

[0205] Results:

[0206] At 2 weeks after operation, the epidural space of the blank group was filled with a large amount of granulation tissue, which extended from the dorsal side to the lateral side of the spinal canal, and reached the nerve root in some cases. The proliferation of granulation tissue and fibroblasts was not active in the polylactic acid membrane group and the experimental group. The matrix and basement membrane were swollen and loose, and there was a small amount of fibroblast and inflammatory cell infiltration. At 4 weeks after operation, the collagen fibers increased in the blank group, and some fibroblasts were transformed into fibrocytes. The number of capillaries decreased, and the collagen fibers were sparse in the polylactic acid membrane group and the experimental group. The composite amniotic membrane material was fused with the fibrous tissue behind it, and the structure was blurred. There was no adhesion between the dura mater and the composite amniotic membrane material. The collagen density was low, and there was no obvious inflammatory reaction. At 8 weeks, the granulation tissue was fibrotic, the collagen was dense in the blank group, the dura mater was adhered to the scar, and bone tissue was formed at the original lamina defect. There was a scar between the dura mater and the new lamina. The collagen density was low in the polylactic acid membrane group, and there was no obvious inflammatory reaction. The epidural scar was small, and there was no adhesion between the dura mater and the polylactic acid membrane. The polylactic acid membrane degraded into small red structureless fragments. At 12 weeks after operation, a large number of collagen fibers were observed in the blank group, which were arranged tightly and chaotically. The cellular components were basically absent, the dura mater was adhered to the scar, and was connected to the new lamina. In the experimental group, the scar area decreased, the cellular components were sparse, and there was no adhesion on the surface of the dura mater under the new bone plate. Some specimens had epidural fat regeneration, and the composite amniotic membrane material was replaced by collagen.

[0207] The microcomputer image processing system calculated the ratio of the intraspinal scar area to the total spinal canal area, measured 3 sections, took their average, and calculated the relative scar index of the spinal canal. Within 2 weeks, the repair of the lamina defect was mainly in the form of granulation tissue, and the collagen fibers were few, and the difference in scar index was not significant (P>0.05). At 4 weeks, the collagen fibers increased, and the scar was initially formed. The experimental group had the smallest value, showing the preventive effect of the composite amniotic membrane material on adhesion (P<0.01). At 8 weeks, the polylactic acid membrane gradually swelled into a gel, and played its role in preventing adhesion, but was still worse than the experimental group (P<0.01). At 12 weeks, the scar index of the experimental group and the polylactic acid membrane group was smaller than that of the blank group (P<0.01), but the difference between the two groups was significant (P<0.05), as shown in the following Table 10:

[0208] Table 10 Comparison of relative scar index at different times (n=5; %)

[0209]

[0210] Comparison between the experimental group and the blank group Comparison with polylactic acid membrane , Statistical analysis was performed by ANOVA analysis method;

[0211] From the above test results, it can be found that the composite amniotic membrane material provided by the application can reduce scar formation, prevent adhesion, and protect exposed nerve roots in spinal surgery.

[0212] The above implementation / test examples are only examples for clearly illustrating, and are not intended to limit the implementation. Based on the above description, other different forms of changes or variations can be made by those skilled in the art. Here, all the implementation is not required or can not be exhausted. The obvious changes or variations derived therefrom are still within the protection scope of the present application.

Claims

1. A composite bioactive membrane material, characterized by: The product comprises a bioactive membrane material and a medical double-sided adhesive tape bonded together. The medical double-sided adhesive tape contains the following components in the indicated mass concentrations and amounts: 30% acrylic acid, 2%–5% collagen-specific binding peptides, 2%–5% elastin-specific binding peptides, 1%–5% isocyanate methacrylate, 1%–5% N-acryloyloxysuccinimide, 1%–5% gelatin methacrylate, 0.5%–5% hydroxypropyl methylcellulose, 0.5%–5% α-ketoglutaric acid, and 0.1%–0.5% magnesium ion compounds, with the remainder being deionized water. The bioactive membrane material is amniotic membrane; The amino acid sequence of the collagen-specific binding peptide is RRKEEDEKED, and the corresponding three-letter amino acid sequence is Arg-Arg-Lys-Glu-Glu-Asp-Glu-Lys-Glu-Asp, with the following structural formula: ； The amino acid sequence of the elastin-specific binding peptide is HHKEDDRRHK, and the corresponding three-letter amino acid sequence is His-His-Lys-Glu-Asp-Asp-Arg-Arg-His-Lys, with the following structural formula: ； Hydroxypropyl methylcellulose is a hydrophilic macromolecule with an average molecular weight of 90 kDa to 120 kDa; Magnesium ion compounds are magnesium chloride or magnesium sulfate.

2. The method of claim 1, wherein the method further comprises the step of: The preparation method of the medical double-sided tape is as follows: acrylic acid, collagen-specific binding peptide, elastin-specific binding peptide, isocyanate methacrylate, N-acryloyloxysuccinimide, gelatin methacrylate, hydroxypropyl methylcellulose, α-ketoglutarate and magnesium ion compound are dissolved in water and mixed evenly. The mixture is then filtered through a 0.4µm sterile syringe filter and poured into a spaced glass mold. The medical double-sided tape is cured in a UV lamp chamber at 20°C~60°C for 55-65 minutes and dried in an oven at 45-55°C for 15-25 minutes. ​ 3. The method for preparing a composite bioactive membrane material according to claim 2, characterized in that: Includes the following steps: (1) Prepare bioactive membrane materials; (2) Preparation of medical double-sided adhesive tape; (3) Apply medical double-sided tape to the outer periphery of the bioactive membrane material, with a coverage area of ​​1 / 16 to 1 / 4 of the bioactive membrane material.

4. The use of a composite bioactive film material according to claim 1, characterized in that: Applications in the preparation of materials for wound management and postoperative repair in ophthalmology, orthopedics, dermatology, and dentistry.

5. Use of a composite bioactive film material according to claim 4, characterized in that: Sutureless amniotic membrane products used in the treatment of ocular surface diseases include medical double-sided tape used to bond the amniotic membrane and bandage lens, preventing the amniotic membrane from slipping off the ocular surface without sutures.

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