CN117679564A - Composite bioactive membrane material, preparation method and application - Google Patents
Composite bioactive membrane material, preparation method and application Download PDFInfo
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- CN117679564A CN117679564A CN202311764618.0A CN202311764618A CN117679564A CN 117679564 A CN117679564 A CN 117679564A CN 202311764618 A CN202311764618 A CN 202311764618A CN 117679564 A CN117679564 A CN 117679564A
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- medical double
- membrane
- adhesive tape
- specific binding
- amniotic membrane
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Abstract
The invention belongs to the technical field of medical materials, and particularly relates to a composite bioactive membrane material, a preparation method and application thereof, wherein the composite bioactive membrane material and a medical double-sided adhesive tape are bonded together, and the medical double-sided adhesive tape comprises the following components in mass concentration and dosage: 30% of acrylic acid, 2% -5% of collagen specific binding peptide, 2% -5% of elastin specific binding peptide, 1% -5% of isocyanoethyl methacrylate, 1% -5% of N-acryloyloxysuccinimide, 1% -5% of gelatin methacrylate, 0.5% -5% of hydroxypropyl methylcellulose, 0.5% -5% of alpha-ketoglutaric acid, 0.1% -0.5% of magnesium ion compound and the balance of deionized water. The composite bioactive membrane material reduces the water absorption expansion rate of the medical double-sided adhesive tape, improves the adhesive force of the bioactive membrane material, and meets the requirement of keeping the adhesive effect for a long time in the local environment with rich water of the wound surface of a human body.
Description
Technical Field
The invention belongs to the technical field of medical materials, and particularly relates to a composite bioactive membrane material, a preparation method and application thereof.
Background
The amniotic membrane is the innermost layer of placenta, contains epithelial cells, is smooth, has no blood vessel, nerve and lymph, has certain elasticity, and has a thickness of about 0.02-0.5 mm. The components of the collagen mainly comprise collagen, fibronectin, laminin, elastin, mucopolysaccharide and the like. Amniotic membrane has wide application in regenerative medicine, for example, as a natural scaffold to facilitate cell growth adhesion, or as a source of various types of stem cells and growth factors.
The amniotic membrane has wide clinical application in wound care, including wound management and postoperative repair in ophthalmology, orthopedics, dermatology and stomatology. The amniotic membrane is shown in an ophthalmic disease related animal experiment and clinical observation: the amniotic membrane can promote the migration of corneal epithelial cells, inhibit the proliferation of new blood vessels, resist inflammation and the like. The amniotic membrane, especially fresh amniotic membrane, contains growth factors which promote epithelialization, facilitate differentiation and migration of epithelial cells and enhance adhesion of epithelial cells. Amniotic membrane prevents leukocyte infiltration and inhibits various proteases such as: trypsin, fibrinogen, collagenase, etc., by inhibiting the corresponding protease, the degree of inflammation is reduced, the duration of inflammation is shortened, and neovascularization is inhibited. In summary, amniotic membrane may be used as a reliable donor material for ophthalmic clinical applications. The amniotic membrane medical instrument product obtained by the traditional Chinese medicine monitoring bureau is in a membrane shape, and diseases suitable for the ophthalmic field comprise pterygium, chemical injury, thermal burn, keratitis, corneal ulcer, blepharocollosis adhesion, glaucoma, bullous keratopathy and the like.
However, the existing amniotic membrane product requires surgical suturing, which on the one hand increases the difficulty of the surgical operation for the doctor and on the other hand causes secondary trauma to the patient in the surgical suturing operation. In order to promote the application of the amniotic membrane product, the trauma to a patient caused by the surgical suture type amniotic membrane graft is avoided, and the suture-free type amniotic membrane material is gradually reported in the literature.
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 of a seamless contact lens type amniotic membrane consisting of a membrane-like amniotic membrane and a soft contact lens using physical molding and non-chemical crosslinking to obtain a basic curvature and requirements similar to those of a soft contact lens for patients with persistent epithelial defects after infectious keratitis. The research results show that: the suture-free contact lens type amniotic membrane does not need surgical suture, and can be used for conveniently and effectively treating patients with continuous epithelial defects after infectious keratitis. However, the amniotic membrane in such products is only physically attached to the soft contact lens substrate, with the risk of weak attachment.
Combining the membrane-like amniotic membrane with medical glue is another direction of 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 (application No. 202080049580.7) disclose a method for preparing a medical double-sided tape for bonding wet tissues and devices, comprising the steps of: 30% (w/w) acrylic acid, 10% (w/w) gelatin, 1% (w/w) N-acryloyloxysuccinimide, 0.1% (w/w) gelatin methacrylate, and 0.2% (w/w) alpha-ketoglutaric acid were dissolved in deionized water. The mixture was then filtered with a 0.4 μm sterile syringe filter and poured onto a glass mold with a space. The medical double-sided tape was cured in an ultraviolet lamp room (284 nm, 10W power) for 20 minutes and dried completely. The final medical double-sided tape was sealed in a plastic bag with desiccant (silicone bag). However, the medical double-sided adhesive tape has the problem of over high water expansion rate (more than 1000%), and the adhesive film sheet-shaped amnion is used for developing and applying the stitching-free amnion, and can generate larger swelling in an environment rich in body fluid (such as an ocular surface), so that the adhesive effect is poor.
The invention discloses a functional tissue engineering material for nerve repair and a preparation method thereof (application number: 201310425493.9), which comprises the following components: a human amniotic membrane as a carrier; neurotrophic factors promoting nerve repair through specific binding of collagen; and immobilizing an inhibitor that binds to calpain. The neurotrophic factors for promoting nerve repair comprise one or more of brain-derived neurotrophic protons, nerve growth factors, neurotrophic factor 3 and glial cell-derived neurotrophic factors; the neurotrophic factor is a fusion protein of the neurotrophic factor and collagen specific binding domain constructed by a genetic engineering method. The gene sequence of the collagen specific binding domain CBD is TKTLRT. The technology only discloses that polypeptide with specific collagen binding domain is overlapped and recombined with other functional factors, and the polypeptide of the specific collagen binding domain only has coordination action binding force with amniotic collagen, and the binding force is weak interaction.
In view of the foregoing, there is a need to develop a composite bioactive membrane material to overcome the defects of the prior art that the water swelling rate of the medical double-sided tape is too high and the adhesion to the amniotic membrane and human tissues is not firm, so as to meet the clinical demands to a greater extent.
Disclosure of Invention
In order to solve the technical problems, the invention provides the composite bioactive membrane material, the preparation method and the application thereof, which reduce the water absorption expansion rate of the medical double-sided adhesive tape, improve the adhesive force to the bioactive membrane material, ensure that the bioactive membrane material meets the requirement of keeping the adhesive effect for a long time in the local environment of the water-rich wound surface of a human body, and further promote the wound healing and the postoperative repair; the index of the water absorption expansion rate of the composite bioactive membrane material is smaller than 300 percent, and the index of the adhesive force is larger than 1.5N after the composite bioactive membrane material is immersed in normal saline for a week, so that the technical problem of infirm adhesion is solved; the preparation method is simple and the cost is low.
The invention solves the technical problems, and the composite bioactive membrane material is characterized in that: the medical double-sided adhesive tape comprises a bioactive film material and a medical double-sided adhesive tape which are bonded together, wherein the medical double-sided adhesive tape comprises the following components in mass concentration and the dosage: 30% of acrylic acid, 2% -5% of collagen specific binding peptide, 2% -5% of elastin specific binding peptide, 1% -5% of isocyanoethyl methacrylate, 1% -5% of N-acryloyloxysuccinimide, 1% -5% of gelatin methacrylate, 0.5% -5% of hydroxypropyl methylcellulose, 0.5% -5% of alpha-ketoglutaric acid, 0.1% -0.5% of magnesium ion compound and the balance of deionized water;
The bioactive membrane material comprises any one of amniotic membrane, small intestine mucosa, pericardium, skin decellularized dressing, chitosan and gelatin.
Wherein the amino acid sequence of the collagen specific binding peptide is RRKEEDEKED, the amino acid sequence corresponding to three letters is Arg-Arg-Lys-Glu-Glu-Asp-Glu-Lys-Glu-Asp, and the structural formula is as follows:
，
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-Arg-Arg-His-Lys, and the structural formula is as follows:
，
the hydroxypropyl methylcellulose is a hydrophilic macromolecule with an average molecular weight of 90-120 kDa;
the magnesium ion compound is magnesium chloride or magnesium sulfate.
The acrylic acid is a hydrophilic polymerizable monomer, and the isocyanoethyl methacrylate is a hydrophobic polymerizable monomer and an amino coupling agent.
The hydroxypropyl methyl cellulose aqueous solution has low temperature sensitivity, and is beneficial to improving the uniformity of the prepared medical double-sided adhesive tape finished product.
The collagen specific binding peptide and the elastin specific binding peptide have the effect of realizing the binding effect with the collagen and the elastin in the local tissues of the human wound surface and the bioactive membrane material, and the binding effect is derived from the receptor-ligand space coordination effect and the electrostatic binding effect between proteins.
The principle of action of the magnesium ion compound is to provide the ionic bond bonding effect among collagen specific binding peptide, elastin specific binding peptide, acrylic acid hydrophilic polymer, bioactive membrane material and local tissues of human wound surface, as shown in a schematic diagram 5.
In the invention, N-acryloyloxy succinimide is taken as an amino coupling agent, gelatin methacrylate is taken as a cross-linking agent, and alpha-ketoglutaric acid is taken as a photoinitiator.
The preparation method of the medical double-sided tape comprises the following steps: dissolving acrylic acid, collagen specific binding peptide, elastin specific binding peptide, isocyanatoethyl methacrylate, N-acryloyloxy succinimide, gelatin methacrylate, hydroxypropyl methylcellulose, alpha-ketoglutaric acid and magnesium ion compound in the rest deionized water, uniformly mixing, filtering the mixture by a 0.4 mu m sterile syringe filter, pouring the mixture into a glass mold with a gap, and curing the medical double-sided adhesive tape in an ultraviolet lamp chamber, namely 365 nm, 85W power and 20-60 ℃ for 55-65 minutes, and drying in a 45-55 ℃ oven for 15-25 minutes.
The preparation method of the composite bioactive membrane material is characterized by comprising the following steps of: the method comprises the following steps:
(1) Preparing a bioactive membrane material;
(2) Preparing a medical double-sided adhesive tape;
(3) And attaching the medical double-sided adhesive tape to the periphery of the bioactive film material, wherein the coverage area of the medical double-sided adhesive tape is 1/16-1/4 of that of the bioactive film material.
The invention relates to application of a composite bioactive membrane material in preparing materials for wound management and postoperative repair of ophthalmology, orthopedics, dermatology and stomatology.
The application of the composite bioactive membrane material can also be applied to a suture-free amniotic membrane product for treating ocular surface diseases, wherein the medical double-sided adhesive tape is used for adhering amniotic membrane and a bandage mirror, and the amniotic membrane is prevented from slipping on the ocular surface under the condition of no suture.
The adhesive suture-free composite bioactive membrane material has the following beneficial effects:
(1) Compared with the prior medical double-sided tape technology (paper: nature, 2019, 575 (7781), 169-174; patent #: 202080049580.7), the water absorption expansion rate is greatly reduced (lower than 300%), so that the adhesive tape meets the requirement of keeping the adhesive effect for a long time (more than one week) in an environment rich in water in vivo. The principle of reducing the water swelling is that the addition of the isocyanoethyl methacrylate hydrophobic monomer reduces the water swelling percentage of the medical double-sided adhesive tape; the addition of collagen specific binding peptides, elastin specific binding peptides, and magnesium compounds increases the binding sites within the tape and thus reduces water penetration.
(2) In terms of the medical double-sided tape adhesion principle, the medical double-sided tape technology disclosed by the invention comprises the following adhesion principles: the hydrophilic polymer of acrylic acid and hydroxypropyl methylcellulose provide hydrogen bond and electrostatic interaction, the N-acryloyloxy succinimide polymer and isocyanoethyl methacrylate polymer provide covalent bond with amino groups in biological tissues, the receptor-ligand space coordination and electrostatic binding between collagen specific binding peptide and elastin specific binding peptide and corresponding proteins provide, and the ionic bonding between collagen specific binding peptide, elastin specific binding peptide, acrylic acid hydrophilic polymer, bioactive membrane material and human wound surface local tissues provide by magnesium ion compound. The four adhesion principles effectively improve the adhesion to biological tissues, and the adhesion is kept to be more than 1.5N after the biological tissues are immersed in normal saline for a week.
(3) The uniformity of the prepared medical double-sided adhesive tape finished product is improved. In contrast to the prior art (paper: nature, 2019, 575 (7781), 169-174; patent # 202080049580.7), aqueous gelatin solutions are very sensitive to temperature, for example, aqueous gelatin pre-prepared solutions with a mass solubility of 10% can solidify at normal temperature (25 ℃), and it is difficult to ensure uniformity of the finished medical double-sided tape. The hydroxypropyl methyl cellulose aqueous solution has low sensitivity to temperature, and is beneficial to improving the uniformity of the prepared medical double-sided adhesive tape finished product.
The adhesive composite bioactive membrane material has wide clinical application in wound care, including wound management and postoperative repair in ophthalmology, orthopedics, dermatology and stomatology.
Drawings
FIG. 1 is a graph showing the results of the water swelling rate test of a medical double-sided tape;
FIG. 2 is a schematic diagram showing a method for testing adhesive force of a double-sided adhesive tape for traditional Chinese medicine according to the present invention;
FIG. 3 is a graph showing the results of adhesive force test of medical double-sided tape;
FIG. 4 is a graph showing the results of a long-term adhesive property test of the medical double-sided tape of the present invention;
( And (3) injection: the left graph shows that the medical double-sided tape seriously absorbs water and swells after the two pigskin sheets are bonded by the double-sided tape in the traditional Chinese medicine of the comparative example 1 are immersed in normal saline for 1 week, and the bonding is not firm and separation occurs; the right figure shows that the two pigskin sheets bonded by the double-sided tape according to the preferred embodiment 2 of the present invention can remain bonded after being immersed in physiological saline for 1 week )
FIG. 5 is a schematic diagram showing the action of magnesium ion compounds in the present invention.
Detailed Description
The invention is further described in connection with the specific embodiments below, wherein membrane sheet amniotic membrane is commercially available. In the embodiments described herein, the "bio-amniotic membrane" is only exemplified as the "bio-amniotic membrane", and as described above, the "bio-amniotic membrane" includes but is not limited to amniotic membrane, intestinal mucosa, pericardium, skin decellularized dressing, chitosan, and gelatin, and the "medical double-sided tape" has adhesive properties to various membrane materials, and does not limit the types of membrane materials.
Example 1:
the method comprises the following steps:
(1) Preparing a medical double-sided adhesive tape;
30% of acrylic acid (the concentrations are mass percentage concentrations), 2% of collagen specific binding peptide (amino acid sequence RRKEEDEKED, purchased from Shanghai Yao Biotechnology Co., ltd.), 2% of elastin specific binding peptide (amino acid sequence HHKEDDRRHK, purchased from Shanghai Yao Biotechnology Co., ltd.), 1% of isocyanatoethyl methacrylate, 1% of N-acryloyloxysuccinimide, 1% of gelatin methacrylate, 0.5% of hydroxypropyl methylcellulose, 0.5% of alpha-ketoglutaric acid and 0.1% of magnesium chloride are dissolved in deionized water. The mixture was then filtered with a 0.4 μm sterile syringe filter and poured onto a glass mold with a space. The medical double-sided tape was cured in an ultraviolet lamp chamber (365, nm, 85W power) at 20 ℃ for 60 minutes and dried in a 50 ℃ oven for 20 minutes. The final medical double-sided tape was sealed in a plastic bag with desiccant (silicone bag).
(2) A medical double-sided tape is locally adhered to one side of the membrane-like amniotic membrane.
The amniotic membrane is from a human. The size of the membrane-like amniotic membrane is 1cm multiplied by 1cm square. Firstly, soaking the membrane-shaped amniotic membrane in sterile physiological saline for more than 5 minutes, keeping the wet state of the membrane-shaped amniotic membrane, and then taking out the amniotic membrane to locally adhere the medical double-sided adhesive tape to the periphery of the membrane-shaped amniotic membrane, wherein the coverage area of the medical double-sided adhesive tape is 1/16 of that of the membrane-shaped amniotic membrane.
Example 2:
the method comprises the following steps:
(1) Preparing a medical double-sided adhesive tape;
30% of acrylic acid (the concentrations are mass percent concentration), 5% of collagen specific binding peptide (amino acid sequence RRKEEDEKED, purchased from Shanghai Yao Biotechnology Co., ltd.), 5% of elastin specific binding peptide (amino acid sequence HHKEDDRRHK, purchased from Shanghai Yao Biotechnology Co., ltd.), 5% of isocyanatoethyl methacrylate, 5% of N-acryloyloxysuccinimide, 5% of gelatin methacrylate, 5% of hydroxypropyl methylcellulose, 5% of alpha-ketoglutaric acid and 0.5% of magnesium chloride are dissolved in deionized water. The mixture was then filtered with a 0.4 μm sterile syringe filter and poured onto a glass mold with a space. The medical double-sided tape was cured in an ultraviolet lamp chamber (365, nm, 85W power) at 20 ℃ for 60 minutes and dried in a 50 ℃ oven for 20 minutes. The final medical double-sided tape was sealed in a plastic bag with desiccant (silicone bag).
(2) A medical double-sided tape is locally adhered to one side of the membrane-like amniotic membrane.
The amniotic membrane is from a human. The membrane sheet amniotic membrane was 3cm×3cm square in size. Firstly, soaking the membrane-shaped amniotic membrane in sterile physiological saline for more than 5 minutes, keeping the wet state of the membrane-shaped amniotic membrane, and then taking out the amniotic membrane to locally adhere the medical double-sided adhesive tape to the periphery of the membrane-shaped amniotic membrane, wherein the coverage area of the medical double-sided adhesive tape is 1/8 of that of the membrane-shaped amniotic membrane.
Example 3:
the method comprises the following steps:
(1) Preparing a medical double-sided adhesive tape;
30% of acrylic acid (the concentrations are mass percent concentration), 3% of collagen specific binding peptide (amino acid sequence RRKEEDEKED, purchased from Shanghai Yao Biotechnology Co., ltd.), 3% of elastin specific binding peptide (amino acid sequence HHKEDDRRHK, purchased from Shanghai Yao Biotechnology Co., ltd.), 3% of isocyanatoethyl methacrylate, 2% of N-acryloyloxysuccinimide, 2% of gelatin methacrylate, 2% of hydroxypropyl methylcellulose, 3% of alpha-ketoglutaric acid and 0.3% of magnesium chloride are dissolved in deionized water. The mixture was then filtered with a 0.4 μm sterile syringe filter and poured onto a glass mold with a space. The medical double-sided tape was cured in an ultraviolet lamp chamber (365, nm, 85W power) at 60 ℃ for 60 minutes and dried in a 50 ℃ oven for 20 minutes. The final medical double-sided tape was sealed in a plastic bag with desiccant (silicone bag).
(2) A medical double-sided tape is locally adhered to one side of the membrane-like amniotic membrane.
The amniotic membrane is from a human. The membrane sheet amniotic membrane was 3cm×3cm square in size. The local adhesion is that firstly, the membrane-shaped amniotic membrane is soaked in sterile normal saline for more than 5 minutes, the wet state of the membrane-shaped amniotic membrane is kept, then the amniotic membrane is taken out, and the medical double-sided adhesive tape is locally adhered to the periphery of the membrane-shaped amniotic membrane, and the coverage area of the medical double-sided adhesive tape is 1/4 of that of the membrane-shaped amniotic membrane.
Example 4:
the method comprises the following steps:
(1) Preparing a medical double-sided adhesive tape;
30% of acrylic acid (the concentrations are mass percent concentration), 5% of collagen specific binding peptide (amino acid sequence RRKEEDEKED, purchased from Shanghai Yao Biotechnology Co., ltd.), 5% of elastin specific binding peptide (amino acid sequence HHKEDDRRHK, purchased from Shanghai Yao Biotechnology Co., ltd.), 5% of isocyanatoethyl methacrylate, 5% of N-acryloyloxysuccinimide, 5% of gelatin methacrylate, 5% of hydroxypropyl methylcellulose, 5% of alpha-ketoglutaric acid and 0.5% of magnesium chloride are dissolved in deionized water. The mixture was then filtered with a 0.4 μm sterile syringe filter and poured onto a glass mold with a space. The medical double-sided tape was cured in an ultraviolet lamp chamber (365, nm, 85W power) at 50 ℃ for 60 minutes and dried in a 50 ℃ oven for 20 minutes. The final medical double-sided tape was sealed in a plastic bag with desiccant (silicone bag).
(2) A medical double-sided tape is locally adhered to one side of the membrane-like amniotic membrane.
The amniotic membrane is derived from pig. The membrane-like amniotic membrane was circular in size with a diameter of 3 cm. The local adhesion is that firstly, the membrane-shaped amniotic membrane is soaked in sterile normal saline for more than 5 minutes, the wet state of the membrane-shaped amniotic membrane is kept, then the amniotic membrane is taken out, and the medical double-sided adhesive tape is locally adhered to the periphery of the membrane-shaped amniotic membrane, and the coverage area of the medical double-sided adhesive tape is 1/8 of that of the membrane-shaped amniotic membrane.
Example 5:
the method comprises the following steps:
(1) Preparing a medical double-sided adhesive tape;
30% of acrylic acid (the concentrations are mass percent concentration), 5% of collagen specific binding peptide (amino acid sequence RRKEEDEKED, purchased from Shanghai Yao Biotechnology Co., ltd.), 5% of elastin specific binding peptide (amino acid sequence HHKEDDRRHK, purchased from Shanghai Yao Biotechnology Co., ltd.), 5% of isocyanatoethyl methacrylate, 5% of N-acryloyloxysuccinimide, 5% of gelatin methacrylate, 5% of hydroxypropyl methylcellulose, 5% of alpha-ketoglutaric acid and 0.5% of magnesium chloride are dissolved in deionized water. The mixture was then filtered with a 0.4 μm sterile syringe filter and poured onto a glass mold with a space. The medical double-sided tape was cured in an ultraviolet lamp chamber (365, nm, 85W power) at 50 ℃ for 60 minutes and dried in a 50 ℃ oven for 20 minutes. The final medical double-sided tape was sealed in a plastic bag with desiccant (silicone bag).
(2) A medical double-sided tape is locally adhered to one side of the membrane-like amniotic membrane.
The amniotic membrane is derived from cattle. The membrane-like amniotic membrane was circular in size with a diameter of 1 cm. The local adhesion is that firstly, the membrane-shaped amniotic membrane is soaked in sterile normal saline for more than 5 minutes, the wet state of the membrane-shaped amniotic membrane is kept, then the amniotic membrane is taken out, and the medical double-sided adhesive tape is locally adhered to the periphery of the membrane-shaped amniotic membrane, and the coverage area of the medical double-sided adhesive tape is 1/8 of that of the membrane-shaped amniotic membrane.
Example 6:
a preparation method of the composite bioactive membrane material is as in example 1, wherein the medical double-sided adhesive tape comprises the following components in mass concentration and dosage: 30% of acrylic acid, 2.2% of collagen specific binding peptide, 3.5% of elastin specific binding peptide, 2.5% of isocyanatoethyl methacrylate, 2.6% of N-acryloyloxysuccinimide, 2.5% of gelatin methacrylate, 3.5% of hydroxypropyl methylcellulose, 4.5% of alpha-ketoglutaric acid, 0.3% of magnesium chloride and the balance of deionized water. The medical double-sided tape was cured in an ultraviolet lamp chamber (365, nm, 85W power) at 45 ℃ for 55 minutes and dried in a 45 ℃ oven for 25 minutes.
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).
The method comprises the following steps:
(1) Preparing a medical double-sided adhesive tape;
30% (w/w) acrylic acid, 10% (w/w) gelatin, 1% (w/w) N-acryloyloxysuccinimide, 0.1% (w/w) gelatin methacrylate, and 0.2% (w/w) alpha-ketoglutaric acid were dissolved in deionized water. The mixture was then filtered with a 0.4 μm sterile syringe filter and poured onto a glass mold with a space. The medical double-sided tape was cured in an ultraviolet lamp room (284 nm, 10W power) for 20 minutes and dried completely. The final medical double-sided tape was sealed in a plastic bag with desiccant (silicone bag).
(2) A medical double-sided tape is locally adhered to one side of the membrane-like amniotic membrane.
The amniotic membrane is from a human. The membrane sheet amniotic membrane was 3cm×3cm square in size. The local adhesion is that firstly, the membrane-shaped amniotic membrane is soaked in sterile normal saline for more than 5 minutes, the wet state of the membrane-shaped amniotic membrane is kept, then the amniotic membrane is taken out, and the medical double-sided adhesive tape is locally adhered to the periphery of the membrane-shaped amniotic membrane, and the coverage area of the medical double-sided adhesive tape is 1/8 of that of the membrane-shaped amniotic membrane.
Comparative example 2:
reference is made to a medical double-sided tape prepared from collagen specific knots and peptides in the patent "functional tissue engineering materials for nerve repair and preparation method thereof" (application number: 201310425493.9).
The method comprises the following steps:
(1) Preparing a medical double-sided adhesive tape;
30% of acrylic acid (the concentrations are mass percentage concentrations), 5% of collagen specific binding peptide (the amino acid sequence is TKTLRT, purchased from Shanghai Yao Biotechnology Co., ltd.), 5% of isocyanatoethyl methacrylate, 5% of N-acryloyloxysuccinimide, 5% of gelatin methacrylate, 5% of hydroxypropyl methylcellulose, 5% of alpha-ketoglutaric acid and 0.5% of magnesium chloride are dissolved in deionized water. The mixture was then filtered with a 0.4 μm sterile syringe filter and poured onto a glass mold with a space. The medical double-sided tape was cured in an ultraviolet lamp chamber (365, nm, 85W power) at 60 ℃ for 60 minutes and dried in a 50 ℃ oven for 20 minutes. The final medical double-sided tape was sealed in a plastic bag with desiccant (silicone bag).
(2) A medical double-sided tape is locally adhered to one side of the membrane-like amniotic membrane.
The amniotic membrane is from a human. The membrane sheet amniotic membrane was 3cm×3cm square in size. The local adhesion is that firstly, the membrane-shaped amniotic membrane is soaked in sterile normal saline for more than 5 minutes, the wet state of the membrane-shaped amniotic membrane is kept, then the amniotic membrane is taken out, and the medical double-sided adhesive tape is locally adhered to the periphery of the membrane-shaped amniotic membrane, and the coverage area of the medical double-sided adhesive tape is 1/8 of that of the membrane-shaped amniotic membrane.
Comparative example 3:
comparative example 3 is a medical double-sided tape prepared with isocyanoethyl methacrylate hydrophobic monomer, but without specific binding peptide and magnesium ion compound.
The method comprises the following steps:
(1) Preparing a medical double-sided adhesive tape;
30% of acrylic acid (the concentrations are mass percent concentration), 5% of isocyanatoethyl methacrylate, 5% of N-acryloyloxysuccinimide, 5% of gelatin methacrylate, 5% of hydroxypropyl methylcellulose and 5% of alpha-ketoglutarate are dissolved in deionized water. The mixture was then filtered with a 0.4 μm sterile syringe filter and poured onto a glass mold with a space. The medical double-sided tape was cured in an ultraviolet lamp chamber (365, nm, 85W power) at 60 ℃ for 60 minutes and dried in a 50 ℃ oven for 20 minutes. The final medical double-sided tape was sealed in a plastic bag with desiccant (silicone bag).
(2) A medical double-sided tape is locally adhered to one side of the membrane-like amniotic membrane.
The amniotic membrane is from a human. The membrane sheet amniotic membrane was 3cm×3cm square in size. The local adhesion is that firstly, the membrane-shaped amniotic membrane is soaked in sterile normal saline for more than 5 minutes, the wet state of the membrane-shaped amniotic membrane is kept, then the amniotic membrane is taken out, and the medical double-sided adhesive tape is locally adhered to the periphery of the membrane-shaped amniotic membrane, and the coverage area of the medical double-sided adhesive tape is 1/8 of that of the membrane-shaped amniotic membrane.
Comparative example 4:
comparative example 4 is a medical double-sided tape prepared with a specific binding peptide and a magnesium compound, but without isocyanatoethyl methacrylate hydrophobic monomer.
The method comprises the following steps:
(1) Preparing a medical double-sided adhesive tape;
30% of acrylic acid (the concentrations are mass percent concentration), 5% of collagen specific binding peptide (amino acid sequence RRKEEDEKED, purchased from Shanghai Yao Biotechnology Co., ltd.), 5% of elastin specific binding peptide (amino acid sequence HHKEDDRRHK, purchased from Shanghai Yao Biotechnology Co., ltd.), 5% of N-acryloyloxy succinimide, 5% of gelatin methacrylate, 5% of hydroxypropyl methylcellulose, 5% of alpha-ketoglutaric acid and 0.5% of magnesium chloride were dissolved in deionized water. The mixture was then filtered with a 0.4 μm sterile syringe filter and poured onto a glass mold with a space. The medical double-sided tape was cured in an ultraviolet lamp chamber (365, nm, 85W power) at 60 ℃ for 60 minutes and dried in a 50 ℃ oven for 20 minutes. The final medical double-sided tape was sealed in a plastic bag with desiccant (silicone bag).
(2) A medical double-sided tape is locally adhered to one side of the membrane-like amniotic membrane.
The amniotic membrane is from a human. The membrane sheet amniotic membrane was 3cm×3cm square in size. The local adhesion is that firstly, the membrane-shaped amniotic membrane is soaked in sterile normal saline for more than 5 minutes, the wet state of the membrane-shaped amniotic membrane is kept, then the amniotic membrane is taken out, and the medical double-sided adhesive tape is locally adhered to the periphery of the membrane-shaped amniotic membrane, and the coverage area of the medical double-sided adhesive tape is 1/8 of that of the membrane-shaped amniotic membrane.
Comparative example 5:
comparative example 5 is a medical double-sided tape containing only the collagen-specific binding peptide described in the present invention, but no elastin-specific binding peptide.
The method comprises the following steps:
(1) Preparing a medical double-sided adhesive tape;
30% of acrylic acid (the concentrations are mass percentage concentrations), 5% of collagen specific binding peptide (amino acid sequence RRKEEDEKED, purchased from Shanghai Yao Biotech Co., ltd.), 5% of isocyanatoethyl methacrylate, 5% of N-acryloyloxysuccinimide, 5% of gelatin methacrylate, 5% of hydroxypropyl methylcellulose, 5% of alpha-ketoglutarate and 0.5% of magnesium chloride are dissolved in deionized water. The mixture was then filtered with a 0.4 μm sterile syringe filter and poured onto a glass mold with a space. The medical double-sided tape was cured in an ultraviolet lamp chamber (365, nm, 85W power) at 60 ℃ for 60 minutes and dried in a 50 ℃ oven for 20 minutes. The final medical double-sided tape was sealed in a plastic bag with desiccant (silicone bag).
(2) A medical double-sided tape is locally adhered to one side of the membrane-like amniotic membrane.
The amniotic membrane is from a human. The membrane sheet amniotic membrane was 3cm×3cm square in size. Firstly, soaking the membrane-shaped amniotic membrane in sterile physiological saline for more than 5 minutes, keeping the wet state of the membrane-shaped amniotic membrane, and then taking out the amniotic membrane to locally adhere the medical double-sided adhesive tape to the periphery of the membrane-shaped amniotic membrane, wherein the coverage area of the medical double-sided adhesive tape is 1/8 of that of the membrane-shaped amniotic membrane.
Comparative example 6:
comparative example 6 is a medical double-sided tape consistent with the composition and process of example 2, but the medical double-sided tape was completely adhered to one side of the membrane-like amniotic membrane, i.e., the medical double-sided tape was 100% completed with one side of the membrane-like amniotic membrane covered.
The technical performance indexes of the medical double-sided tape disclosed by the invention are compared with those of the existing medical double-sided tape technology, and the following table is provided:
table 1 technical comparison table
| Existing medical double-sided adhesive tape Techniques for 1 Features and limitations | The technical characteristics and changes of the medical double-sided tape Feeding in | |
| In an exemplary embodiment Component (A) | Acrylic acid, gelatin, N-propyl Alkene acyloxy succinylidene Amine, gelatin methacrylic acid Esters, alpha-ketoglutarate | Acrylic acid, collagen specific binding peptide and elastic egg White specific binding peptides, isocyanoethyl methacrylate Esters, N-acryloyloxy succinimides, gelatin methyl groups Acrylic ester, hydroxypropyl methyl cellulose and alpha-ketoglutarate Acid and magnesium ion compounds |
| Index of water expansion rate 2 | More than 1000% | Less than 300% |
| Reducing water swelling Principle of | --- | (1) Addition of isocyanoethyl methacrylate hydrophobic monomer Reduces the water absorption expansion percentage of the medical double-sided adhesive tape A rate; (2) Collagen specific binding peptides, elastin Increased addition of specific binding peptides and magnesium compounds The adhesive force of the adhesive tape is improved, and the water infiltration is reduced. |
| Principle of adhesion | (1) Acrylic hydrophilic polymer Compound and gelatin extractFor supplying Hydrogen bonding and electrostatic interactions Using; (2) N-acryloyloxy Polymerization of an sulfoximine Object-provided and biological group Amino incorporation in the weave results Is a covalent bond of (c). | (1) Acrylic hydrophilic polymer and hydroxypropyl methyl fiber Hydrogen bonding and electrostatic interactions provided by the vitamins; (2) N- Acryloyloxy succinimide polymers and methylpropane Isocyanoethyl enoate polymers provided and biocomponents Covalent bonds resulting from the binding of amino groups in the fabric; (3) Collagen egg White specific binding peptides, elastin specific binding peptides Provided a receptor-ligand space between the corresponding protein An inter-coordination and electrostatic bonding; (4) Magnesium ions Collagen specific binding peptides, bullets provided by the compounds Hydrophilic polymerization of specific binding peptides and acrylic acid for sexual proteins Ion between the living things, amniotic membrane and local tissue of human wound surface Bond bonding. |
| Immersing in physiological saline Post-day adhesion index 3 | Less than 0.05N | Greater than 1.5N |
| Stability of the prefabricated solutions | Temperature of aqueous gelatin solution Very sensitive, e.g. of mass Gelatin water-soluble with 10% solubility The liquid is at normal temperature (25 DEG) Will solidify the bottom and is difficult to protect Medical double-sided for syndrome preparation Uniformity of tape finish | Temperature sensitivity of aqueous hydroxypropyl methylcellulose solutions Lower, is favorable for improving the prepared medical double-sided adhesive tape Uniformity of the product. |
( And (3) injection: the "existing medical double-sided tape technology" refers to paper: nature, 2019, 575 (7781), 169-174, and patent#: 202080049580.7; the water absorption expansion index is data obtained by the method in the test example 1 medical double-sided adhesive tape water absorption expansion test; the "adhesive force index after one day immersion in physiological saline" is data obtained by the method of the "adhesive force test of medical double-sided tape of test example 2" according to the present invention )
Test example 1 Water absorption expansion Rate test of medical double-sided tape
The test contained the following materials: examples 1-5 and comparative examples 1-5. Examples 1-5 are, among other things, medical double-sided tapes prepared by the method described in the present invention. 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). Comparative example 2 is a medical double-sided tape prepared from collagen-specific junction and peptide in reference patent "functional tissue engineering material for nerve repair and preparation method thereof" (application number: 201310425493.9). Comparative example 3 is a medical double-sided tape prepared with isocyanoethyl methacrylate hydrophobic monomer, but without specific binding peptide and magnesium ion compound. Comparative example 4 is a medical double-sided tape prepared with a specific binding peptide and a magnesium compound, but without isocyanatoethyl methacrylate hydrophobic monomer. Comparative example 5 is a medical double-sided tape containing only the collagen-specific binding peptide described in the present invention, but no elastin-specific binding peptide.
The testing method comprises the following steps:
the oven-dried medical double-sided tape was weighed and immersed in physiological saline at 37℃in an oven for one week. After each removal, the surface was wiped with filter paper to remove excess liquid and then reweighed. The water expansion rate was calculated by the following formula:
，
Ww and Wd represent the mass of the wet and dry samples, respectively, after one week of soaking.
The experimental results are shown in table 2 and fig. 1:
TABLE 2
The experimental results show that the medical double-sided tape of examples 1-5 has a significantly reduced water swelling rate (from over 900% to below 300%) compared to the medical double-sided tape of comparative examples 1-5. Comparative example 2 is a medical double-sided tape prepared from collagen-specific junction and peptide in the reference patent "functional tissue engineering material for nerve repair and preparation method thereof" (application number: 201310425493.9), because the technical principle only considers the coordination binding force of collagen-specific junction and peptide with human amniotic collagen, the binding force has very small effect on inhibiting water swelling (the water swelling rate is still up to 1022%).
The effect of reducing the water swelling rate was very weak (from 1115% to 945% and 978%) by means of the technology of reducing the water swelling rate in single comparative example 3 (addition of isocyanatoethyl methacrylate hydrophobic monomer to reduce the water swelling percentage of medical double-sided tape) or comparative example 4 (addition of collagen specific binding peptide, elastin specific binding peptide, magnesium ion compound to increase binding points inside the tape, and thus less penetration of water).
The medical double-sided tape of examples 1 to 5 showed a significantly reduced water swelling rate (from 912% to 300% or less) compared to comparative example 5 (medical double-sided tape containing only the collagen-specific binding peptide of the present invention but no elastin-specific binding peptide).
The sequence design of the collagen specific binding peptide and the elastin specific binding peptide in the medical double-sided adhesive tape system further improves the ionic bond effect between the collagen specific binding peptide and the elastin specific binding peptide and magnesium ion compounds, improves chemical crosslinking points in the internal network structure of the adhesive tape, and achieves the effect of greatly reducing the water absorption expansion rate; in the invention, the corresponding specific binding peptide is independently and optimally designed aiming at two protein compositions, namely collagen and elastin, which account for the main proportion in tissues such as amniotic membrane, human eye surface and the like, so that the coordination acting force is further improved. The superposition of the two technical principles in the invention has the unexpected technical effect of greatly reducing the water absorption expansion rate (lower than 300 percent).
Test example 2 adhesion test of medical double-sided tape
The test contained the following materials: examples 1-5 and comparative examples 1-5. Examples 1-5 are, among other things, medical double-sided tapes prepared by the method described in the present invention. 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). Comparative example 2 is a medical double-sided tape prepared from collagen-specific junction and peptide in reference patent "functional tissue engineering material for nerve repair and preparation method thereof" (application number: 201310425493.9). Comparative example 3 is a medical double-sided tape prepared with isocyanoethyl methacrylate hydrophobic monomer, but without specific binding peptide and magnesium ion compound. Comparative example 4 is a medical double-sided tape prepared with a specific binding peptide and a magnesium compound, but without isocyanatoethyl methacrylate hydrophobic monomer. Comparative example 5 is a medical double-sided tape containing only the collagen-specific binding peptide described in the present invention, but no elastin-specific binding peptide.
The testing method comprises the following steps: fresh pigskin was cut to 1cm by 3cm strips and medical double-sided tape was cut to 1cm by 1cm strips. After wetting the surface of the pigskin with physiological saline, pressing the medical double-sided adhesive tape with the length of 1cm multiplied by 1cm to one end of the smooth surfaces of the two pigskins for 1 minute, immersing the pigskin in the physiological saline for 1 day, taking out the pigskin, and clamping the other ends of the two pigskins by adopting a clamp of a tensile tester. The maximum tensile force is recorded as the adhesive force of the medical double-sided adhesive tape.
Fig. 2 is a schematic diagram showing a method for testing the adhesive force of the double-sided adhesive tape for traditional Chinese medicine according to the present invention.
The experimental results are shown in table 3 and fig. 3:
TABLE 3 Table 3
Experimental results show that compared with the medical double-sided tape prepared by the method in comparative examples 1-5, the medical double-sided tape in examples 1-5 effectively improves the adhesive force and can meet the requirement of keeping the adhesive effect in the environment with the eye surface rich in water.
As can be seen from table 3 above, if compared with the single effects of comparative example 2 (collagen-specific junction and peptide), comparative example 3 (isocyanoethyl methacrylate hydrophobic monomer), and comparative example 4 (specific binding peptide and magnesium ion compound), the effect of improving the adhesive force under rich water environment in the present invention is very remarkable, and the requirement that the adhesive force can be effectively maintained after one day of immersion in physiological saline is fulfilled.
The medical double-sided tape of examples 1-5 showed a greater improvement in adhesion (from 0.32N to 1.5N or more) compared to comparative example 5 (medical double-sided tape containing only the collagen-specific binding peptide of the present invention, but no elastin-specific binding peptide).
From the whole technical point of view, the water absorption expansion percentage of the medical double-sided adhesive tape is reduced by combining two principles of reducing the water absorption expansion in the invention (1) adding the isocyano ethyl methacrylate hydrophobic monomer; (2) The addition of the collagen specific binding peptide, the elastin specific binding peptide and the magnesium ion compound improves the binding points inside the adhesive tape so as to improve the penetration of less water, and the combination of four binding principles, namely hydrogen bond and electrostatic interaction provided by the acrylic hydrophilic polymer and the hydroxypropyl methyl cellulose; an N-acryloyloxy succinimide polymer and an isocyanoethyl methacrylate polymer provide covalent bonds with amino groups in biological tissues; collagen-specific binding peptides, elastin-specific binding peptides, and receptor-ligand steric coordination with the corresponding proteins; the specific binding peptide of the collagen, the specific binding peptide of the elastin, the acrylic acid hydrophilic polymer and the ionic bond binding function between the amniotic membrane and local tissues of the wound surface of the human body provided by the magnesium ion compound can realize the requirement that the adhesive force can be effectively maintained after the human body is immersed in physiological saline for 1 day, and the effect on the adhesive force is very obvious.
Test example 3 long-term adhesive property test of medical double-sided tape
The test contained 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).
The testing method comprises the following steps: fresh pigskin and medical double-sided tape were cut to 1cm by 3cm strips. After wetting the surface of the pigskin with physiological saline, the medical double-sided tape is pressed to the smooth surfaces of the two pigskins, and then the two bonded pigskins are soaked in the physiological saline for 1 week in a baking oven at 37 ℃. And then taking out, observing and photographing, and recording the adhesion state of the medical double-sided adhesive tape to the pigskin.
The experimental results are shown in fig. 4: the two pigskin sheets adhered by the double-sided tape in the traditional Chinese medicine of comparative example 1 are immersed in normal saline for 1 week, and the medical double-sided tape is seriously swelled by water absorption and is not firmly adhered to generate separation. The two pigskin sheets bonded with the double-sided tape according to the preferred embodiment 2 of the present invention can remain bonded after immersing them in physiological saline for 1 week.
Test example 4 test of long-term adhesion Property by means of partial adhesion and Whole adhesion
The test contained the following materials: preferred examples 2 and 6. Comparative example 6 is a medical double-sided tape consistent with the composition and process of example 2, but the medical double-sided tape was completely adhered to one side of the membrane-like amniotic membrane, i.e., the medical double-sided tape was 100% completed with one side of the membrane-like amniotic membrane covered.
The testing method comprises the following steps: the medical double-sided tape and the membrane-shaped amniotic membrane bonded in example 2 and comparative example 6 were immersed in physiological saline in an oven at 37 ℃ for 1 week, respectively, and then taken out for observation and recording of the bonding state of the two.
The experimental results show that: with the preferred example 2 of the present invention, the medical double-sided tape and the membrane-like amniotic membrane remained adhered after 1 week of immersion, but the medical double-sided tape and the membrane-like amniotic membrane of comparative example 6 were separated after 1 week of immersion. The medical double-sided tape and the membrane-shaped amniotic membrane are proved to be better than the complete bonding mode in terms of keeping bonding force in an underwater environment, because the medical double-sided tape can be extruded with the membrane-shaped amniotic membrane after being expanded by water absorption in the complete bonding mode, and further the medical double-sided tape and the membrane-shaped amniotic membrane are easy to separate, namely the bonding time is limited.
Test example 5 application of composite amniotic membrane material in ophthalmic surgery
Establishing an animal model: taking 20 New Zealand rabbits (2+/-0.5 kg), carrying out intravenous injection anesthesia on the rabbits by using sodium pentobarbital, fixing the upper side of the right eye, removing villi and eyelashes around the right eye by using a shaver, and carrying out disinfection treatment by using iodophor. Local anesthesia of ocular surface with obucaine, aspiration of excessive water from cornea with dry filter paper, infiltration of H at 1mol/L 2 SO 4 A single layer of circular filter paper of about 6 a mm a diameter was applied to the surface of the central region of the cornea and the filter paper was removed for 30 seconds. Excess acid solution on the cornea was removed by suction with a dry filter paper, and then rinsed with a large amount of physiological saline for 5 min. After one week of molding, the wound healing condition is observed, the model of the cornea full layer with light burn and self-healing and overweight burn is removed, and the moderate and light burn is taken. 16 models with self-healing ability that did not allow wound healing were used for the healing experiments.
The operation method comprises the following steps: the operation is carried out by using sterile surgical instruments and strictly according to the clinical operation requirements.
(1) Adhesive suture-free composite amniotic membrane material experimental group
Randomly taking 8 burn models (2+/-0.5 kg), adopting sodium pentobarbital for intravenous injection for anesthesia, fixing the front and rear limbs by ropes, wrapping the whole body of an experimental rabbit by an operation hole towel, removing the villus around the right eye of the rabbit by an electric shaver, and sterilizing by using iodophor to reduce the influence on the operation process and the later cornea repair as much as possible. The operation part is a wound area of a corneal acid burn model, firstly, the damaged part is debrided under a microscope, and necrotic corneal epithelium and tissues are thoroughly cleaned by a jewel knife. The adhesive composite amniotic membrane material prepared in the preferred embodiment is spread and covered on the whole cornea surface, and is pressed for 1 minute.
(2) Blank control group
The rest 8 burn models are taken, the operation method is the same as that of the experimental group, the adhesive composite amniotic membrane material is not used, and the rest operation and the later care are the same as those of the experimental group.
Post-operative evaluation: the following evaluation items were observed and photographed:
(1) The mental state and the movement of the experimental rabbits are observed every day, and the cornea repair condition is observed.
(2) Animal experiment evaluation criteria: overall, the morphology after cornea repair was observed, and cornea repair in animal experiments was evaluated according to the following table classification, as shown in table 4 below:
TABLE 4 Table 4
( And (3) injection: referring to the index standard of national eye trauma occupational eye disease group and the grading of Roper-Hall on the degree of eye chemical injury, the evaluation index is 0-1 grade and 2-4 grade is invalid, the single item is invalid when evaluating the overall validity, namely, the judgment is invalid, and all data are statistically analyzed after evaluation )
(3) The corneal turbidity and the corneal neovascularization of the blank control group and the experimental group were observed at 1 week, 4 weeks and 8 weeks after the operation by the above-mentioned evaluation method of the "animal experiment evaluation standard". The blank control and experimental groups were stained with sodium fluorescein at 4 weeks and 8 weeks to observe the area of corneal epithelial defects. The safety and effectiveness of the adhesive composite amniotic membrane material for repairing the ocular surface are comprehensively evaluated.
Analysis of results:
(1) Post-operative vascularization scoring results table 5 below:
TABLE 5
(2) Post-operative scarring scoring results, table 6 below:
TABLE 6
(3) Post-operative epithelialization scoring results, table 7 below:
TABLE 7
The results of the comparative study on the effectiveness of the amniotic membrane group and the blank group by using the rabbit corneal acid burn model show that the adhesive composite amniotic membrane material experimental group provided by the invention can better assist in repairing the moderate and mild acid burn of the cornea.
Test example 6 application of adhesive suture-free composite amniotic membrane material in skin defect
And selecting 12 healthy New Zealand white rabbits of the pure breed of the international standard experimental animal, wherein the male and female animals are not limited, and the weight is 2.5-3.0 kg. Randomization was divided into 2 treatment groups: experimental group implanted the sample prepared in the preferred example 2 of the present invention, and the control group was a vaseline yarn group.
The specific experimental steps are as follows:
the back of the patient is dehaired by 8% sodium sulfide solution before operation, and after the successful anesthesia of 2% pentobarbital sodium (30 mg/kg) intraperitoneal injection, the skin of the back operation field is disinfected and spread with towel. 2X 2cm square full-layer skin defects are manufactured on two sides of the back midline, the left side is an experimental group, the experimental group is flatly paved on the whole cornea surface, and the cornea surface is pressed for 30 seconds. The right side is a control group, and the wound surface is covered with vaseline oil yarn. After the operation is finished, the materials are wrapped and fixed.
Post-operation observation: and (3) observing wound healing conditions after operation by 1W, 2W, 3W and 4W, measuring the size of a wound, and calculating the wound healing rate. Post-operative 4W harvest healing zone tissue was observed pathologically.
Wound healing rate = healing area/original area x 100
The results are shown in Table 8 below:
table 8 comparison of wound healing rates in experimental and control groups
From the results, the adhesive composite amniotic membrane material provided by the invention can accelerate wound tissue healing compared with the traditional oily yarn.
Test example 7 application of adhesive suture-free composite amniotic membrane material in tendon repair
And selecting 60 pure healthy New Zealand white rabbits of the international standard experimental animals, wherein the male and female animals are not limited, and the weight is 2.5-3.0 kg. Randomization into 3 treatment groups: experimental group was implanted with the sample prepared in the preferred example 2 of the present invention, and the control group 1 was a polylactic acid film group control group 2 and a blank control group.
The specific experimental steps of the rabbit toe tendon animal model include:
(1) Experimental grouping
Experimental group: taking 20 New Zealand white rabbits (2+/-0.5 kg) and carrying out intravenous injection anesthesia by using sodium pentobarbital, placing the white rabbits into a fixed box, stretching the left limbs of the rabbits out of the fixed box, using a shaver to disinfect the left hind limbs Mao Chujin of the rabbits by using iodophor. The surgical site is rabbit middle finger flexor tendon, a surgical knife is used for making a longitudinal incision at the surgical site to find out the flexor tendon, after the flexor tendon is lifted by a vascular forceps, the defect is cut by the operation (the width is about 1/2 of the tendon), the defective tendon is sutured by adopting a Bunnell suturing mode, the used suture is 5-0 single-strand polypropylene suture, and the sutured site is wrapped by adopting the sample prepared in the preferred embodiment 2 of the invention. Finally, the operation limb is fixed for 3 weeks by gypsum.
Polylactic acid film group: the surgical method of taking 20 New Zealand white rabbits is the same as that of experimental groups, and the tendon is sutured, the suture part is wrapped by a polylactic acid anti-adhesion film, and then peripheral wounds are sutured and gypsum is fixed.
Blank control group: the operation method of taking 20 New Zealand white rabbits is the same as that of experimental groups, except that the tendon is directly sutured to the peripheral wound after suturing, and gypsum is fixed.
The above operations are all performed by using sterile surgical instruments, and are strictly performed according to clinical operation requirements. The experimental animals were given cefazolin (40 mg/day) for antibacterial and carborphen (4 mg/kg) for pain relief within 3 days after surgery. The mental state and the activity of the experimental rabbits were observed daily.
(2) Postoperative material sampling
The plaster of all test rabbits is removed 3 weeks after operation, the free movement condition of the test rabbits, especially the movement condition of the operation limbs, and the operation toes are manually pulled, so that the expansion resistance of the toes is felt in the pulling process, and the expansion resistance is obviously different from that of normal toes.
And 10 rabbits are randomly selected from an experimental group, a polylactic acid membrane group and a blank control group for sampling and observing after 3 weeks and 6 weeks after operation, an operation part is firstly scratched by an operation blade, and the adhesion condition of tendons is generally observed. Animals were sacrificed by intravenous air injection at the ear margin of 20mL, surgical tendons were immediately removed, and each group of samples was subjected to a tensile strength test using an electronic universal tester (model: RGM-6002T), and tensile force was applied to the samples at a speed of 5 mm/min. After the sample is destroyed, the computer automatically outputs the elastic modulus data of each group of specimens, and the biomechanical characteristics of the tendons are evaluated.
(3) Tendon biomechanical characterization results
The tensile test analysis results show that the elastic modulus of tendons of the test group and the polylactic acid film group and the blank control group at each observation time point are obviously improved. At the 3 rd week after operation, the tendon elastic modulus (maximum breaking load) of the experimental group is obviously better than that of the polylactic acid membrane group and the blank control group (P < 0.05); the elastic modulus of the samples of the experimental group and the polylactic acid film group and the blank control group are not significantly different at the 6 th week after operation. The amniotic membrane can effectively improve the tensile strength of injured tendons in early repair, and the experimental group shows the best tendon repair effect, and the following details are shown in table 9:
TABLE 9 tendon biomechanical characterization results
The experimental group is respectively compared with the polylactic acid film group and the blank control group;。
test example 8 application of composite amniotic membrane material in spinal surgery
60 adult New Zealand rabbits are selected, and the weight of the adult New Zealand rabbits is 2.0-3.0 kg, so that the male and female rabbits are not restricted. According to the differences of the epidural coverings at the vertebral plate defects, 60 white rabbits are randomly divided into an experimental group, a polylactic acid membrane group and a blank control group, and 20 white rabbits are respectively divided into each group.
The length of the median incision after taking under aseptic operation is about 20mm, the L5 spinous process is cut layer by layer, the L5 vertebral plate is bitten by a miniature vertebral plate rongeur, the exposed area of the dura mater of 10mm multiplied by 5mm is caused, and the epidural fat is removed. After complete hemostasis, the experimental group (sample prepared in preferred example 2 in the present invention) and the polylactic acid film were covered outside the dura mater, respectively, according to the pre-operation random grouping, without any intervening covering group as a blank control group. All animals were kept in separate cages under the same conditions after surgery without taking any medicine.
The content and method are observed:
each group of specimens was collected and treated by 5 animals each at 2, 4, 8 and 12 weeks post-surgery, and the L5 spinal column including sacrospinous muscle and vertebral body appendages was completely removed after animal sacrifice, and paraffin-embedded sections were performed after formalin fixation, with a thickness of 5 μm. Tissue sections were stained for HE, masson and picric acid-sirius scarlet, respectively.
Observing the distribution of the epidural scar and the relation between the epidural scar and nerve roots under a common optical microscope by using an optical microscope;
computer image analysis (axioplan 2 image, usa) calculates the scar cross-sectional area of the irregular morphology and the ratio of the scar cross-sectional area to the spinal canal area is used for scar index determination.
Results:
through observation of a common microscope, a large amount of granulation tissues are filled in defects outside the dura mater of the postoperative 2-week blank group, extend from the back side to the side of the vertebral canal, and reach the root of the nerve. The polylactic acid membrane group and the experimental group have the advantages that granulation tissues and fibroblasts are not actively proliferated, the matrix and the basement membrane of the experimental group are swollen and loose, and a small amount of fibroblasts and inflammatory cells infiltrate. The 4 weeks blank group after operation has increased collagen fibers, partial fibroblasts are converted into fibroblasts, capillaries are reduced, the polylactic acid membrane group and experimental composition have fewer fibroblasts, collagen fibers are rare, and epidural gaps exist. The composite amniotic membrane material is fused with the fiber tissue at the rear of the composite amniotic membrane material, has a fuzzy structure and is not adhered with the dura mater; the collagen density is low, and no obvious inflammatory reaction exists. At 8 weeks, the granulation tissue was fibrosed, the blank group was dense in collagen, the dura and scar were adhered, and bone tissue was formed at the original lamina defect, with scar between it and the dura. The polylactic acid membrane group has low collagen density and no obvious inflammatory reaction; less epidural scar and no adhesion with the dura; the polylactic acid film is degraded into red-dyed unstructured small fragments. A large amount of collagen fibers can be seen in the space-time white group 12 weeks after operation, and the arrangement is compact and disordered; the cell components are basically disappeared, and the dura mater is adhered tightly with the scar and connected with the new vertebral plate. The scar area is reduced after 8 weeks and 12 weeks of operation of the experimental group, the cell components are rare, the surface of the dura mater under the new bone plate is not adhered, part of specimens have epidural fat regeneration, and the composite amniotic membrane material is replaced by collagen.
The microcomputer image processing system calculates the ratio of the scar area in the invasive vertebral canal to the whole vertebral canal area, measures 3 slices, takes the average value of the slices, and calculates the relative scar index in the vertebral canal. The vertebral plate defect was repaired within 2 weeks with granulation tissue forming predominantly, less collagen fibers, and insignificant scar index differences (P > 0.05). Collagen fibers are increased at 4 weeks, scars are initially formed, and the numerical value of the experimental group is minimum, so that the composite amniotic membrane material has the effect of preventing adhesion (P < 0.01). The polylactic acid film gradually swells into a gel at 8 weeks, exerting its blocking-preventing properties, but still worse than the experimental group (P < 0.01). The scar index was smaller at 12 weeks for both the experimental and polylactic acid film groups than for the blank group (P < 0.01), but the difference between the two groups was significant (P < 0.05), see table 10 below:
table 10 comparison of relative scar index at different times (n=5;%)
Comparison of experimental group with blank groupThe method comprises the steps of carrying out a first treatment on the surface of the Compared with polylactic acid film->，/>The method comprises the steps of carrying out a first treatment on the surface of the The statistical analysis adopts an ANOVA analysis method;
from the test results, the composite amniotic membrane material provided by the invention can reduce scar formation and prevent adhesion in spinal surgery, and has a protective effect on exposed nerve roots.
The above examples/experiments are only examples for clarity of illustration and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.
Claims (5)
1. A composite bioactive membrane material is characterized in that: the medical double-sided adhesive tape comprises a bioactive film material and a medical double-sided adhesive tape which are bonded together, wherein the medical double-sided adhesive tape comprises the following components in mass concentration and the dosage: 30% of acrylic acid, 2% -5% of collagen specific binding peptide, 2% -5% of elastin specific binding peptide, 1% -5% of isocyanoethyl methacrylate, 1% -5% of N-acryloyloxysuccinimide, 1% -5% of gelatin methacrylate, 0.5% -5% of hydroxypropyl methylcellulose, 0.5% -5% of alpha-ketoglutaric acid, 0.1% -0.5% of magnesium ion compound and the balance of deionized water;
the bioactive membrane material comprises any one of amniotic membrane, small intestine mucosa, pericardium, skin decellularized dressing, chitosan and gelatin;
wherein the amino acid sequence of the collagen specific binding peptide is RRKEEDEKED, the amino acid sequence corresponding to three letters is Arg-Arg-Lys-Glu-Glu-Asp-Glu-Lys-Glu-Asp, and the structural formula is as follows:
，
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-Arg-Arg-His-Lys, and the structural formula is as follows:
，
the hydroxypropyl methylcellulose is a hydrophilic macromolecule with an average molecular weight of 90-120 kDa;
The magnesium ion compound is magnesium chloride or magnesium sulfate.
2. The method for preparing the composite bioactive membrane material according to claim 1, wherein the method comprises the following steps: the preparation method of the medical double-sided tape comprises the following steps: dissolving acrylic acid, collagen specific binding peptide, elastin specific binding peptide, isocyanoethyl methacrylate, N-acryloyloxy succinimide and gelatin methacrylate, uniformly mixing hydroxypropyl methylcellulose, alpha-ketoglutaric acid and magnesium ion compound in water, filtering the mixture by a 0.4 mu m sterile syringe filter, pouring the mixture into a glass mold with intervals, curing the medical double-sided adhesive tape for 55-65 minutes in an ultraviolet lamp room at 20-60 ℃, and drying the medical double-sided adhesive tape in an oven at 45-55 ℃ for 15-25 minutes.
3. The method for preparing the composite bioactive membrane material according to claim 2, wherein the method comprises the following steps: the method comprises the following steps:
(1) Preparing a bioactive membrane material;
(2) Preparing a medical double-sided adhesive tape;
(3) And attaching the medical double-sided adhesive tape to the periphery of the bioactive film material, wherein the coverage area of the medical double-sided adhesive tape is 1/16-1/4 of that of the bioactive film material.
4. The use of a composite bioactive film as claimed in claim 1, wherein: application of the material in preparing wound management and postoperative repair materials for ophthalmology, orthopedics, dermatology and stomatology.
5. The use of a composite bioactive film as claimed in claim 4, wherein: the stitching-free amniotic membrane product is applied to the treatment of ocular surface diseases, wherein the medical double-sided adhesive tape is used for adhering the amniotic membrane and a bandage mirror, and the amniotic membrane is prevented from slipping on the ocular surface under the condition of no suture.
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