CN106310385A - Preparation method and application of egg shell membrane composite nano-silver thin film used for artificial skin - Google Patents
Preparation method and application of egg shell membrane composite nano-silver thin film used for artificial skin Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/60—Materials for use in artificial skin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
- A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
- A61L15/40—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing ingredients of undetermined constitution or reaction products thereof, e.g. plant or animal extracts
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
- A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
- A61L15/42—Use of materials characterised by their function or physical properties
- A61L15/46—Deodorants or malodour counteractants, e.g. to inhibit the formation of ammonia or bacteria
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/54—Biologically active materials, e.g. therapeutic substances
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/10—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing inorganic materials
- A61L2300/102—Metals or metal compounds, e.g. salts such as bicarbonates, carbonates, oxides, zeolites, silicates
- A61L2300/104—Silver, e.g. silver sulfadiazine
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/404—Biocides, antimicrobial agents, antiseptic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/80—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special chemical form
Abstract
The invention discloses a preparation method and application of an egg shell membrane composite nano-silver thin film used for an artificial skin. The preparation method comprises the following steps: removing egg white and yolks of fresh eggs and washing egg shells with de-ionized water; separating egg shell membranes and washing the egg shell membranes with the de-ionized water; cutting the egg shell membranes into sizes of (1cm-3cm)*(1cm-3cm) and putting the egg shell membranes into a hydrochloric acid solution with the concentration of 1mM to 100mM and the pH (Potential of Hydrogen) of 1 to 3; immersing for 1 to 4 days at room temperature and washing with the de-ionized water; then putting the egg shell membranes into a dopamine solution with the concentration of 2mg/mL to 4mg/mL and the pH of 8.5 to 9.5; immersing for 6h to 20h at room temperature and washing with the de-ionized water; then putting the egg shell membranes into a silver nitrate solution with the concentration of 10nM to 50nM; immersing for 6h to 20h at room temperature without light, and then washing with the de-ionized water; and naturally drying at room temperature to obtain the egg shell membrane composite nano-silver thin film used for the artificial skin. The prepared egg shell membrane composite nano-silver thin film is used for manufacturing the artificial skin, has excellent mold resistance, and has no obvious biological toxicity; and the preparation method is simple in process, less in consumed time and low in cost.
Description
Technical field
The invention belongs to materials application technical field, be specifically related to a kind of eggshell membrane composite nano silver for artificial skin thin
The preparation method and application of film.
Background technology
Skin, as the organ of human body maximum, can protect internal from damage and to maintain water-electrolyte balance.
The skin injury that burn, wound, diabetic foot ulcer etc. cause, can cause body occur serious infection, Water-Electrolyte is unbalance,
The problems such as scar hyperplasia, bring the painful and heavy medical burden of huge mind & body to patient.Therefore, skin is recovered
Barrier function become the task of top priority.But, the patient that partial skin defect is bigger, such as large area burn and scald patient, due to from
Body skin source is limited, it is impossible to timely and effective wound closure.For solving this problem, a kind of suitably artificial skin of application substitutes certainly
Body skin source wound closure is the most necessary.
At present, the artificial skin of the most many commercializations such as Integra, AlloDerm is applied to clinic.Integra
It is in a kind of artificial skin of exploitation in 1980 by Burke and Yannas.It is made up of " epidermis ", glutaraldehyde silicon rubber film
The cattle NTx of crosslinking constitutes " corium " with 6-chondroitin sulfate and glucosamine.Clinical practice finds that it is conducive to wound surface
The infiltration growth of tissue, it is easy to vascularization and the most degradable, can effectively facilitate the healing of large-area burn wound, meanwhile,
Preventing and treating keloid and hypertrophic cicatrix also there is positive effect.But, wound surface is required higher by this artificial skin, infection energy
Power is weak, and mechanical strength and permeability the most poor, simultaneously as complicated process of preparation, expensive, it is difficult to extensively push away
Extensively.AlloDerm is then a kind of commercialization acellular dermal matrix produced by Life Science company of the U.S., and it is by newly
The artificial skin that the epidermis of fresh cadaver skin and cell component are made after removing, because eliminating cell component, so AlloDerm
Reduce immunogenicity, but its three dimensional structure still remaining extracellular matrix support and complete basement membrane complex,
The growth extension of bootable regenerative cell.But, owing to cadaver skin source is limited, and have the danger of transmitted virus, simultaneously AlloDerm
Expensive, complex process, its application is also restrained.
Therefore, preparation a kind of cheap, operating procedure is relatively easy, and the people that good biocompatibility, antibacterial effect are good
Work skin is particularly important.
The performance of timbering material is the key factor building artificial skin, is also the study hotspot in this field.Eggshell membrane, be
A kind of fibrous web-like thin film between Ovum crusta Gallus domesticus and Ovum Gallus domesticus album, has good permeability and mechanical characteristic.Eggshell membrane master
To be made up of the protein such as keratin, collagen protein, to constitute extracellular matrix possibly together with hyaluronic acid, chondroitin sulfate etc. simultaneously
Main component.As a kind of natural biological timbering material, it has good biological tissue's compatibility, and nontoxic, without exempting from
Epidemic focus.Additionally, eggshell membrane is easily obtained, cheap, the most extensively can apply with simple economy ground.At present, it has been
Through the treatment for the shallow skin wound of some table.
Nanometer silver is a kind of widely used medical antibacterial agent, has good killing effect to antibacterial, fungus etc..With
Time, nanometer silver antimicrobial is continuous and stable, rare drug resistance.Research is it has proven convenient that nanometer silver can effectively facilitate wound by antimicrobial antiphlogistic
Heal in face.Therefore, the antibiotic preparation that nanometer silver can be effective as in artificial skin plays a role.But, nanometer silver is at sky
Silver oxide it is easily formed with oxygen so that it is antibacterial efficacy is substantially reduced and even disappears after gas contacts.Recent study finds, by nanometer
Silver is incorporated on biological support and can effectively keep its stability and antibiotic property.
Prepare nanometer silver at present and be integrated into biological support to prepare the method for artificial skin more, including electrostatic spinning
Technology, original reagent etc. is gone back in application.But, electrostatic spinning technique operation is relative complex, relatively costly, and conventional original reagent of going back needs
Time more and be easily generated environmental contaminants.In recent years, Messersmith finds that dopamine can be the surface of various materials oneself
Polymerization forms a layers of polymer dopamine thin film, and this thin film can adhere to different kinds of molecules or cell efficiently.And it is polymerized dopamine
There is reproducibility, in situ silver ion reduction can be become nano-Ag particles at material surface.Research confirms, this kind of nanometer silver has
Good stability and antibiotic property.Additionally, dopamine is nontoxic, without teratogenesis, there is good biological tissue's compatibility, permissible
It is efficiently applied to the preparation of artificial skin.
To sum up, with eggshell membrane as biological support, utilizing adhesion and the reduction characteristic of dopamine, a kind of eggshell membrane of preparation is combined
The antibacterial artificial skin of the nanometer silver method that to be that one facilitates feasible, simple and practical.
Summary of the invention
It is an object of the invention to provide the preparation method of a kind of eggshell membrane composite Nano Ag films for artificial skin.This
The anti-microbial property of the eggshell membrane composite Nano Ag films for artificial skin of invention preparation is good, without obvious bio-toxicity, and can
Effectively facilitating the reparation of full thickness dermal wounds, its preparation method has that technique is simple, time-consuming less, the advantage of low cost.
The present invention provides the preparation method of a kind of eggshell membrane composite Nano Ag films for artificial skin, including walking as follows
Rapid:
(1) take Fresh Egg, discard Ovum Gallus domesticus album and egg yolk, Ovum crusta Gallus domesticus is placed in deionized water cleaning, separate eggshell membrane, then spend
After ionized water cleans, eggshell membrane is cut into 1 3cm × 1 3cm size；
(2) eggshell membrane that step (1) prepares being placed in concentration is 1 100mM, in the hydrochloric acid solution of pH=1 3, soaks under room temperature
14 days, then clean 35 times with deionized water, every all over 35 minutes；
(3) being dissolved in by dopamine in the Tris HCl buffer that concentration is 10 100mM, prepared concentration is that 2 4mg/mL are many
Bar amine aqueous solution, by pH regulator to 8.5 9.5；
(4) eggshell membrane that step (2) prepares is placed in the dopamine solution that step (3) prepares, soaks 6 20h under room temperature, so
Clean 35 times with deionized water afterwards, every all over 35 minutes；
(5) eggshell membrane that step (4) prepares being placed in concentration is in 10 50nM silver nitrate solutions, and under room temperature, lucifuge soaks 6
20h, then cleans 35 times with deionized water, every all over 35 minutes, and natural drying under room temperature prepares eggshell membrane composite nano silver
Thin film.
Further, the eggshell membrane deionized water cleaning that described step (1), step (2) and step (4) prepare 3 times, every time
5 minutes.
Further, the concentration of the hydrochloric acid solution of described step (2) is 1mM, pH=3.
Further, the Tris HCl buffer concentration of described step (3) is 10mM.
Further, the dopamine solution concentration that described step (3) prepares is 2mg/mL, pH=8.5.
Further, the concentration of the silver nitrate solution of described step (5) is 30nM.
A kind of eggshell membrane composite Nano Ag films for artificial skin is used for treating the artificial skin of skin injury in preparation
Purposes in skin or surgical dressing.
Further, described skin injury is skin full-thickness defects or skin infection wound surface.
The eggshell membrane composite Nano Ag films of the present invention is carried out following performance test.
1. scanning electron microscopic observation:
See Fig. 1, use scanning electron microscope to observe the table of prepared eggshell membrane composite Nano Ag films under different multiples
Face structure.Use Image-Pro Plus 6.0 software, measure the porosity of all electron microscopic picture thin film, export to Excel table
In lattice, calculate porosity meansigma methods and standard deviation obtains: the porosity of thin film is 45 ± 4.11%.
2. transmission electron microscope observing:
See Fig. 2 and Fig. 3, prepared eggshell membrane composite Nano Ag films Ultrasonic Pulverization instrument pulverized, is dissolved in deionized water,
Piping and druming is formed uniformly suspension, takes a suspension and drops on the coated copper mesh of charcoal, uses the chicken that transmission electron microscope observation prepares
The form of nanometer silver and size in egg film composite Nano Ag films.Using Image-J software, arranging scale is legend scale, Gu
Surely select this scale, measure all nano-Ag particles diameters, export in Excel form, calculate nanometer silver size distribution figure, ginseng
See Fig. 3, and try to achieve meansigma methods: a diameter of 13.91nm of nanometer silver.
The invention has the beneficial effects as follows: compared with prior art, the present invention, with eggshell membrane as biological support, utilizes dopamine
Adhesion and reduction characteristic, the antibiotic property of nanometer silver, prepare a kind of eggshell membrane composite Nano Ag films for artificial skin, tool
There is good anti-microbial property, without obvious bio-toxicity；Preparation method low cost, time-consumingly few, operating procedure is simple.
Accompanying drawing explanation
Fig. 1 is the electron scanning micrograph of the eggshell membrane composite Nano Ag films of one embodiment of the invention.
Fig. 2 is the transmission electron microscope photo of the eggshell membrane composite Nano Ag films of one embodiment of the invention.
Fig. 3 be the eggshell membrane composite Nano Ag films of one embodiment of the invention transmission electron microscope photo in nanometer
The size distribution figure of silver.
Fig. 4 is the cytoactive block diagram in the cell compatibility experiment of the eggshell membrane composite Nano Ag films prepared.
Fig. 5 shows anti-microbial property test experience result (a aseptic filter paper of the eggshell membrane composite Nano Ag films of preparation
Eggshell membrane after sheet, b HCl treatment, the eggshell membrane of c dopamine solution process, d eggshell membrane composite Nano Ag films).
Fig. 6 shows that zoopery is using the eggshell membrane after blank group, kpetrolatum gauze, HCl treatment, many respectively
Eggshell membrane and eggshell membrane composite Nano Ag films after the process of bar amine aqueous solution process the 3rd day and the 7th day wounds in mice after wound surface
The comparison of healing.
Detailed description of the invention
Embodiment 1: the preparation method of a kind of eggshell membrane composite Nano Ag films for artificial skin, including walking as follows
Rapid:
(1) take Fresh Egg, after breaking into pieces gently, discard Ovum Gallus domesticus album and egg yolk, Ovum crusta Gallus domesticus is placed in deionized water cleaning, then uses
Tweezers careful separation eggshell membrane, after again cleaning with deionized water, is cut into 2cm × 2cm size by eggshell membrane；
(2) eggshell membrane that step (1) prepares is placed in hydrochloric acid solution, soaks under room temperature 3 days, then clean 3 with deionized water
Time, every all over 5 minutes, the concentration of described hydrochloric acid solution is 1mM, pH=3；
(3) being dissolved in by dopamine in Tris HCl solution, described Tris HCl solution concentration is 10mM, and prepared dopamine is molten
Liquid concentration is 2mg/ml, pH regulator to 8.5；
(4) eggshell membrane that step (2) prepares is placed in the dopamine solution that step (3) prepares, soaks 16h under room temperature, then
Clean 3 times with deionized water, every all over 5 minutes；
(5) being placed in silver nitrate solution by the eggshell membrane that step (4) prepares, the concentration of described silver nitrate (5) solution is 30nM, room
The lower lucifuge of temperature soaks 18h, then cleans 3 times with deionized water, every all over 5 minutes, and natural drying under room temperature prepares eggshell membrane and is combined
Nano silver film.
Embodiment 2: the preparation method of a kind of eggshell membrane composite Nano Ag films for artificial skin, including walking as follows
Rapid:
(1) take Fresh Egg, after breaking into pieces gently, discard Ovum Gallus domesticus album and egg yolk, Ovum crusta Gallus domesticus is placed in deionized water cleaning, then uses
Tweezers careful separation eggshell membrane, after again cleaning with deionized water, is cut into 1cm × 3cm size by eggshell membrane；
(2) eggshell membrane that step (1) prepares is placed in hydrochloric acid solution, soaks under room temperature 1 day, then clean 3 with deionized water
Time, every all over 5 minutes, the concentration of described hydrochloric acid solution is 10mM, pH=1；
(3) being dissolved in by dopamine in Tris HCl solution, described Tris HCl solution concentration is 30mM, and prepared dopamine is molten
Liquid concentration is 2mg/ml, pH regulator to 9.0；
(4) eggshell membrane that step (2) prepares is placed in the dopamine solution that step (3) prepares, soaks 6h under room temperature, then use
Deionized water cleans 3 times, every all over 5 minutes；
(5) being placed in silver nitrate solution by the eggshell membrane that step (4) prepares, the concentration of described silver nitrate (5) solution is 10nM, room
The lower lucifuge of temperature soaks 6h, then cleans 3 times with deionized water, every all over 5 minutes, and natural drying under room temperature prepares eggshell membrane and is combined
Nano silver film.
Embodiment 3: the preparation method of a kind of eggshell membrane composite Nano Ag films for artificial skin, including walking as follows
Rapid:
(1) take Fresh Egg, after breaking into pieces gently, discard Ovum Gallus domesticus album and egg yolk, Ovum crusta Gallus domesticus is placed in deionized water cleaning, then uses
Tweezers careful separation eggshell membrane, after again cleaning with deionized water, is cut into 3cm × 3cm size by eggshell membrane；
(2) eggshell membrane that step (1) prepares is placed in hydrochloric acid solution, soaks under room temperature 4 days, then clean 5 with deionized water
Time, every all over 3 minutes, the concentration of described hydrochloric acid solution is 100mM, pH=3；
(3) being dissolved in by dopamine in Tris HCl solution, described Tris HCl solution concentration is 100mM, prepared dopamine
Solution concentration is 4mg/ml, pH regulator to 9.5；
(4) eggshell membrane that step (2) prepares is placed in the dopamine solution that step (3) prepares, soaks 20h under room temperature, then
Clean 5 times with deionized water, every all over 3 minutes；
(5) being placed in silver nitrate solution by the eggshell membrane that step (4) prepares, the concentration of described silver nitrate (5) solution is 50nM, room
The lower lucifuge of temperature soaks 20h, then cleans 5 times with deionized water, every all over 3 minutes, and natural drying under room temperature prepares eggshell membrane and is combined
Nano silver film.
Embodiment 4: the preparation method of a kind of eggshell membrane composite Nano Ag films for artificial skin, including walking as follows
Rapid:
(1) take Fresh Egg, after breaking into pieces gently, discard Ovum Gallus domesticus album and egg yolk, Ovum crusta Gallus domesticus is placed in deionized water cleaning, then uses
Tweezers careful separation eggshell membrane, after again cleaning with deionized water, is cut into 2cm × 3cm size by eggshell membrane；
(2) eggshell membrane that step (1) prepares is placed in hydrochloric acid solution, soaks under room temperature 2 days, then clean 4 with deionized water
Time, every all over 4 minutes, the concentration of described hydrochloric acid solution is 40mM, pH=2；
(3) being dissolved in by dopamine in Tris HCl solution, described Tris HCl solution concentration is 50mM, and prepared dopamine is molten
Liquid concentration is 3mg/ml, pH regulator to 8.5；
(4) eggshell membrane that step (2) prepares is placed in the dopamine solution that step (3) prepares, soaks 14h under room temperature, then
Clean 4 times with deionized water, every all over 4 minutes；
(5) being placed in silver nitrate solution by the eggshell membrane that step (4) prepares, the concentration of described silver nitrate (5) solution is 40nM, room
The lower lucifuge of temperature soaks 12h, then cleans 4 times with deionized water, every all over 4 minutes, and natural drying under room temperature prepares eggshell membrane and is combined
Nano silver film.
Embodiment 5: the preparation method of a kind of eggshell membrane composite Nano Ag films for artificial skin, including walking as follows
Rapid:
(1) take Fresh Egg, after breaking into pieces gently, discard Ovum Gallus domesticus album and egg yolk, Ovum crusta Gallus domesticus is placed in deionized water cleaning, then uses
Tweezers careful separation eggshell membrane, after again cleaning with deionized water, is cut into 3cm × 2cm size by eggshell membrane；
(2) eggshell membrane that step (1) prepares is placed in hydrochloric acid solution, soaks under room temperature 3 days, then clean 5 with deionized water
Time, every all over 4 minutes, the concentration of described hydrochloric acid solution is 80mM, pH=3；
(3) being dissolved in by dopamine in Tris HCl solution, described Tris HCl solution concentration is 70mM, and prepared dopamine is molten
Liquid concentration is 4mg/ml, pH regulator to 9.5；
(4) eggshell membrane that step (2) prepares is placed in the dopamine solution that step (3) prepares, soaks 10h under room temperature, then
Clean 5 times with deionized water, every all over 4 minutes；
(5) being placed in silver nitrate solution by the eggshell membrane that step (4) prepares, the concentration of described silver nitrate (5) solution is 40nM, room
The lower lucifuge of temperature soaks 14h, then cleans 5 times with deionized water, every all over 4 minutes, and natural drying under room temperature prepares eggshell membrane and is combined
Nano silver film.
Embodiment 6: prepared eggshell membrane composite Nano Ag films is carried out the use that cell compatibility experiment is inspection preparation
In the cell compatibility of the eggshell membrane composite Nano Ag films of artificial skin, test as follows:
(1) experiment packet: experimental group: eggshell membrane composite Nano Ag films；Matched group: the eggshell membrane after HCl treatment and dopamine
The eggshell membrane processed；
(2) the experimental group sample prepared by embodiment 1 and matched group sample are made with card punch respectively the thin film of a diameter of 6mm
Disk, rinses 3 times with phosphate buffer after being soaked in 75% ethanol solution sterilization；
(3) aseptic disk is respectively placed in 96 orifice plates, often group multiple cropping 6 hole；
(4) take the logarithm trophophase GFP l cell, every hole inoculation 8 × 103Cell, on thin film disk, adds containing 10%
The DMEM culture medium of hyclone, to 200 μ L, changes liquid after 24 hour cell adherent growth；
(5) cultivate after the 3rd day, take the thin film often organizing each 3 holes under fluorescence microscope observation of cell adhere to and growthform；
Remaining 3 holes and carry out cell proliferation test experience, discard former culture medium, every hole adds 100 culture medium new for μ L and the MTT of 10 μ L
Reagent, hatches at 37 DEG C 4 hours, then discards culture medium, adds 100 μ l dimethyl sulfoxide and ties with the purple on dissolving films
Crystalline substance, after shaking table rocks 10 minutes, utilizes microplate reader to detect the optical density in every hole at 570nm wavelength, draws block diagram, detection
The impact on cytoactive of the eggshell membrane composite nano silver.
Result: see Fig. 4, under mirror, visible three groups of cells all grow preferably, launch in typical fusiformis, and cytoactive inspection
Surveying the eggshell membrane group no significant difference that display eggshell membrane composite nano silver processes with dopamine, the addition of prompting nanometer silver does not causes
Substantially cytotoxicity, safety is preferable.After dopamine processes, eggshell membrane does not soaks the eggshell membrane of dopamine and is obviously promoted cell
Growth, illustrates that dopamine has good biocompatibility.
Embodiment 7: prepared eggshell membrane composite Nano Ag films carried out anti-microbial property test experience
For the anti-microbial property of the eggshell membrane composite Nano Ag films for artificial skin of inspection preparation, application disk diffusion method enters
The following experiment of row:
(1) experiment packet: experimental group: the eggshell membrane composite Nano Ag films that embodiment 1 prepares；Matched group: aseptic filter paper sheet salt
Eggshell membrane after acid treatment, the eggshell membrane after dopamine solution process；
(2) by method in embodiment 6, each cluster film is made aseptic disk；
(3) bacterial suspension of escherichia coli and staphylococcus aureus is diluted to 0.5 standard Maxwell unit (106cfu/
Ml), then dipping bacterium solution with aseptic cotton carrier and be spread evenly across respectively on MH agar plates, every kind of bacterial strain repeats 3；
(4) respectively each cluster film disk is carefully placed on MH agar plates, and culture dish is put in 37 DEG C of incubators；
Take out culture dish after (5) 24 hours, take pictures and record.
Result: see Fig. 5, in escherichia coli and staphylococcus aureus culture dish, eggshell membrane composite nano silver is thin
The surrounding of film all occurs in that obvious antibacterial ring, illustrates that it is respectively provided with good antibacterial effect to two kinds of antibacterials.
Embodiment 8；Prepared eggshell membrane composite Nano Ag films is carried out full thickness dermal wounds reparative experiment
(1) preparation of BALB/C mice (purchased from Third Military Medical University's Experimental Animal Center) full thickness dermal wounds model: real
Test animal and give 1% pentobarbital sodium lumbar injection (100mg/kg, 0.01ml/g) anesthesia.Mice is fixed on ventricumbent position, back art
After district's preserved skin, conventional 75% ethanol disinfection.Respectively make the full thickness dermal wounds of an a diameter of 4mm in both sides, back with card punch.
Take each 7 mices of BALB/C mice 35, experimental group and matched group, prepare wound model the most as stated above.
(2) experimental group: eggshell membrane composite nano silver film sample embodiment 1 prepared is cut to 6 × 6mm size, nothing
After bacterium normal saline flushing, it is covered on mouse back wound surface, then is fixed on skin of back with vacuum suction film；Matched group: point
Not Yong eggshell membrane flap coverage after eggshell membrane, dopamine process after kpetrolatum gauze, HCl treatment, then consolidate with vacuum suction film
Fixed, and blank group is only fixed with vacuum suction film.
(3) postoperative Taking Pictures recording immediately, and as the original wound surface area of the 0th day, then opened each group little in the 3rd, 7 days
Mus wound surface checks healing state, and Taking Pictures recording.After postoperative 7th day takes pictures, mice i.e. uses disconnected cervical approach to put to death.
(4) utilize Image Pro Plus 6.0 respectively to organize wound surface photo to the 3rd, 7 days to be analyzed, calculate residual wound face
Long-pending (area that edge of wound is surrounded), and it is calculated as follows Wound healing rate:
Wound healing rate=(original wound surface area-residual wound area)/original wound surface area.
Result: see Fig. 6, eggshell membrane composite Nano Ag films group wound surface has no and is showed no redness and allergic phenomena, without oozing
Going out liquid, pus and abscess to be formed, art district tissue has no congested, edema and necrosis.Postoperative 3rd day, eggshell membrane composite nano silver was thin
Relatively remaining respectively organizes Wound healing rate height to film group, and has significant difference compared with blank group and kpetrolatum gauze group；Postoperative
7th day, eggshell membrane composite Nano Ag films group healing rate was the highest compared with other four groups, and has significant difference.This result explanation
The eggshell membrane composite Nano Ag films for artificial skin prepared can remarkably promote wound healing.
Claims (8)
1. the preparation method for the eggshell membrane composite Nano Ag films of artificial skin, it is characterised in that include walking as follows
Rapid:
(1) take Fresh Egg, discard Ovum Gallus domesticus album and egg yolk, Ovum crusta Gallus domesticus is placed in deionized water cleaning, separate eggshell membrane, then spend
After ionized water cleans, eggshell membrane is cut into 1 3cm × 1 3cm size；
(2) eggshell membrane that step (1) prepares being placed in concentration is 1 100mM, in the hydrochloric acid solution of pH=1 3, soaks under room temperature
14 days, then clean 35 times with deionized water, every all over 35 minutes；
(3) being dissolved in by dopamine in the Tris HCl buffer that concentration is 10 100mM, prepared concentration is that 2 4mg/mL are many
Bar amine aqueous solution, by pH regulator to 8.5 9.5；
(4) eggshell membrane that step (2) prepares is placed in the dopamine solution that step (3) prepares, soaks 6 20h under room temperature, so
Clean 35 times with deionized water afterwards, every all over 35 minutes；
(5) eggshell membrane that step (4) prepares being placed in concentration is in 10 50nM silver nitrate solutions, and under room temperature, lucifuge soaks 6
20h, then cleans 35 times with deionized water, every all over 35 minutes, and natural drying under room temperature prepares eggshell membrane composite nano silver
Thin film.
The preparation method of a kind of eggshell membrane composite Nano Ag films for artificial skin the most according to claim 1, its
It is characterised by that the eggshell membrane deionized water that described step (1), step (2) and step (4) prepare cleans 3 times, every all over 5 minutes.
The preparation method of a kind of eggshell membrane composite Nano Ag films for artificial skin the most according to claim 1, its
The concentration being characterised by the hydrochloric acid solution of described step (2) is 1mM, pH=3.
The preparation method of a kind of eggshell membrane composite Nano Ag films for artificial skin the most according to claim 1, its
The Tris HCl buffer concentration being characterised by described step (3) is 10mM.
The preparation method of a kind of eggshell membrane composite Nano Ag films for artificial skin the most according to claim 1, its
It is characterised by that the dopamine solution concentration that described step (3) prepares is 2mg/mL, pH=8.5.
The preparation method of a kind of eggshell membrane composite Nano Ag films for artificial skin the most according to claim 1, its
The concentration being characterised by the silver nitrate solution of described step (5) is 30nM.
7. the eggshell membrane composite Nano Ag films for artificial skin prepared by the method described in claim 16 any bar
In preparation purposes in the artificial skin treating the skin injury of people or surgical dressing.
Purposes the most according to claim 7, described skin injury is skin full-thickness defects or skin infection wound surface.
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| CN116440330A (en) * | 2023-04-12 | 2023-07-18 | 山东第一医科大学附属省立医院(山东省立医院) | Multifunctional anti-adhesion biological film and preparation method and application thereof |
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