CN115869453A - Double-layer antibacterial dressing loaded with antibacterial molecules, preparation and application - Google Patents
Double-layer antibacterial dressing loaded with antibacterial molecules, preparation and application Download PDFInfo
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- CN115869453A CN115869453A CN202111128441.6A CN202111128441A CN115869453A CN 115869453 A CN115869453 A CN 115869453A CN 202111128441 A CN202111128441 A CN 202111128441A CN 115869453 A CN115869453 A CN 115869453A
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention provides a double-layer antibacterial dressing loaded with antibacterial molecules, and preparation and application thereof. The double-layer antibacterial dressing comprises: the fish skin gelatin nano fiber layer loaded with antibacterial molecules and the acellular fish skin matrix layer are characterized in that the thickness of the fish skin gelatin nano fiber layer is 0.05-2mm, the diameter of the fish skin gelatin nano fiber is 100-600nm, and the thickness of the acellular fish skin matrix layer is 0.01-1mm. From the structure and the components, the acellular fish skin contains more abundant fat and omega-3 fatty acid, can obviously promote the generation of new vessels in a wound area, can effectively induce wound repair, has low immunogenicity and good biological safety, has low risk of spreading viruses of zoonosis, can not cause autoimmune reaction of an organism, can provide a barrier effect for an inner acellular fish skin matrix layer by combining with a fish skin gelatin nanofiber layer containing antibacterial peptide, reduces the influence of the external environment, can better play a role in repairing tissues, and can further improve the antibacterial activity of a double-layer dressing by adding the antibacterial peptide.
Description
Technical Field
The invention relates to the technical field of preparation of biomedical materials. More particularly, relates to a double-layer antibacterial dressing loaded with antibacterial molecules, and preparation and application thereof.
Background
Burn is one of the most painful and difficult diseases in all the diseases at present. The existing burn treatment mostly adopts a mode of matching modern medical science and technology means with medicines, but the method of sacrificing skin for life can still enable partial patients with large-area burn to survive. Although researchers have developed active epidermal repair tissues, they are still in the first stage of application.
The acellular matrix is an acellular tissue slice made of animal or human skin, reserves the collagen component and the tissue basic structure of dermis and the basement membrane component, is the most ideal cell microenvironment at present, becomes a hotspot for the development and result transformation of bionic regenerative medical products, and is applied to the fields of wound and burn repair, oral medicine, tissue defect and the like. However, the prior art still has the following problems: (1) Burn wounds are often accompanied by excessive exudate, and the wounds have a large amount of bacterial biofilm production rate due to the fact that the wound contains a large amount of organic substances which are beneficial to bacterial growth, so that the wound healing difficulty is aggravated, and currently, fish skin gelatin hardly has a bacteriostatic effect. (2) The acellular fish skin matrix has a loose three-dimensional structure, is difficult to isolate from the external environment, and is easy to cause wound infection.
Therefore, the development of a novel antibacterial dressing which has the advantages of antibacterial effect, isolation effect, regular structure and industrial production and can effectively promote the healing of burn wounds is urgently needed.
Disclosure of Invention
The invention aims to provide a double-layer antibacterial dressing loaded with antibacterial molecules, which has the advantages of antibiosis, high biological safety, strong wound repair capacity, good isolation effect, regular structure and the like.
The second purpose of the invention is to provide a preparation method for preparing the double-layer antibacterial dressing, which is simple to operate, cheap and easily available in raw materials and capable of realizing industrial production.
A third object of the present invention is to provide a use of a bi-layer antimicrobial dressing as described above.
In order to achieve the first purpose, the invention adopts the following technical scheme:
the invention provides a double-layer antibacterial dressing loaded with antibacterial molecules, which comprises: a fish skin gelatin nanofiber layer and a decellularized fish skin matrix layer which are loaded with antibacterial molecules; wherein the thickness of the fish skin gelatin nanofiber layer is 0.05-2mm, the diameter of the fish skin gelatin nanofiber is 100-600nm, and the thickness of the acellular fish skin matrix layer is 0.01-1mm.
The invention provides an antibacterial dressing with a double-layer structure, wherein the outer layer of the antibacterial dressing is a fish skin gelatin nanofiber layer loaded with antibacterial molecules, the inner layer of the antibacterial dressing is a decellularized fish skin matrix layer, and from the aspects of structure and components, the outer fish skin gelatin nanofiber layer can provide a barrier effect for the inner layer of the decellularized fish skin matrix layer, so that the influence of the external environment is reduced, the decellularized fish skin matrix layer is effectively protected, the tissue repairing effect is better exerted, and the antibacterial activity of the double-layer dressing can be further improved by adding antibacterial peptide; the inner layer of the acellular fish skin has a bionic structure similar to that of a pig and a cow leather, contains more abundant fat and omega-3 fatty acid, can remarkably promote the generation of new blood vessels in a wound area, has a remarkable promoting effect on wound repair, particularly the repair of a difficult wound, can more effectively induce the repair of the wound, has low immunogenicity of an acellular fish skin matrix and good clinical use safety, has a lower risk of spreading viruses of zoonosis than the pig and the cow leather from a land mammal, and cannot cause the autoimmune reaction of an organism.
Further, the antibacterial molecule-loaded fish skin gelatin nanofiber layer is formed on the acellular fish skin matrix layer through an electrostatic spinning technology.
Further, the antimicrobial molecule comprises an antimicrobial peptide.
Further, the antimicrobial peptide includes, but is not limited to, one or more of insect antimicrobial peptide, mammal antimicrobial peptide, amphibian antimicrobial peptide, fish antimicrobial peptide, mollusk antimicrobial peptide, crustacean antimicrobial peptide, plant antimicrobial peptide or bacterial antimicrobial peptide.
The antibacterial peptide has the characteristics of strong alkali resistance, good thermal stability, broad-spectrum antibiosis and the like, is mainly introduced to improve the antibacterial activity of the double-layer antibacterial dressing, prevent the wound repairing effect from being influenced by the invasion of external bacteria in the wound repairing process, and simultaneously inhibit the bacterial breeding of the wound.
In order to achieve the second purpose, the invention adopts the following technical scheme:
the invention provides a preparation method for preparing the double-layer antibacterial dressing, which comprises the following steps:
dissolving fish skin enzymolysis gelatin in water, preparing an electrostatic spinning precursor solution with antibacterial molecules, carrying out electrostatic spinning on the acellular fish skin matrix layer by using an electrostatic spinning technology to form a fish skin gelatin nanofiber layer, and carrying out hot-pressing lamination, crosslinking and drying to obtain the double-layer antibacterial dressing.
In the invention, the fish skin gelatin nanofiber layer is uniformly covered on the acellular fish skin matrix layer by controlling the process parameters of the electrostatic spinning technology, so that the double-layer antibacterial dressing with a regular structure is obtained, and the double-layer antibacterial dressing can be cut into regular geometric figures such as rectangles according to specific application requirements, and the specification of the dressing can be changed according to the size of a wound surface, for example: 5cm × 5cm, 5cm × 10cm or 10cm × 10cm. The outer layer fish skin gelatin nanofiber not only has good barrier effect on external bacteria and dust, but also effectively protects the fish skin matrix layer and plays a role in repairing tissues; the outer layer consisting of the nano-fibers has good and quick imbibition performance, and can discharge effusion of the wound quickly and in time; in addition, the addition of the antibacterial peptide can promote the nano-fiber to play a role in lasting and efficient antibacterial in the degradation process.
In the application, after the hot-pressing bonding is placed in the electrostatic spinning, the acellular fish skin matrix and the fish skin gelatin nanofiber layer are better compounded together, and the crosslinking is to realize the controllable degradation and continuous antibiosis of the double-layer antibacterial dressing.
The term fish skin enzymatic gelatin as used herein refers to a fish skin enzymatic gelatin prepared by subjecting a fish skin gelatin to an enzymatic hydrolysis process, wherein the enzymatic hydrolysis process may be performed using existing well-established techniques.
Further, the concentration of the fish skin enzymolysis gelatin in water is 5-15%; in the electrostatic spinning precursor solution, the concentration of antibacterial molecules is 0.5-1%; the temperature for preparing the electrostatic spinning precursor solution is 45-60 ℃. In the specific embodiment of the present invention, preferably, the concentration of the fish skin enzymatic gelatin is 10%; the temperature for preparing the electrostatic spinning precursor solution is 50 ℃.
Further, the voltage of the electrostatic spinning is 15-25 kV, and the jet distance of the syringe needle of the electrostatic spinning is 7-25cm.
Further, the hot-pressing laminating temperature is 40-60 ℃; the hot-pressing laminating time is 0.1-30 s.
Further, the cross-linking agent selected for cross-linking includes but is not limited to one or more of glutaraldehyde, genipin, formaldehyde, epichlorohydrin and sodium trimetaphosphate.
Further, the concentration of the crosslinking agent is 1 to 5%.
Further, the crosslinking time is 24-48 h.
Further, the preparation method also comprises sterilization after the drying is finished.
Further, the sterilization is performed by one of high-pressure steam sterilization, co60 sterilization, electron beam irradiation sterilization and ethylene oxide sterilization.
Further, the sterilization adopts electron beam irradiation for sterilization.
Furthermore, the irradiation dose of the electron beam irradiation sterilization is 10-20 KGy, and the irradiation time is 0.1-7 s.
The preparation of the acellular fish skin matrix layer can refer to the method in the prior art, and aims to prepare the complete acellular fish skin matrix with a three-dimensional structure by degreasing and acellular treatment of the outsourcing fish skin, and the invention provides a method for preparing the acellular fish skin matrix layer, which comprises the following steps:
removing scales, fish flesh and inner fascia from fresh fish skin, soaking in saturated sodium chloride aqueous solution, treating with acid, cleaning with alkali solution, repeatedly freezing and thawing at-60 deg.C, taking out, removing cells with surfactant, washing with alkali solution, and freeze drying to obtain cell-removed fish skin matrix layer.
Further, the acid solution for acid treatment includes but is not limited to one or more of acetic acid solution, citric acid solution, phosphoric acid solution or formic acid solution, and the acid solution concentration is 1-3%.
Further, the alkali solution includes, but is not limited to, one or more of sodium hydroxide, potassium hydroxide, calcium hydroxide or ammonia water; the concentration of the alkali liquor is 3-5%.
In a specific embodiment of the present invention, the acid solution has a concentration of 2%; the concentration of the alkali liquor is 4%.
Further, the surfactant includes, but is not limited to, one or more of sodium dodecyl sulfate, tween 80, sodium deoxycholate, or Triton X-100. Wherein the concentration of the surfactant is 0.3-0.5%.
In order to achieve the third purpose, the invention adopts the following technical scheme:
the invention provides an application of the double-layer antibacterial dressing or the double-layer antibacterial dressing prepared by the preparation method in preparing a product for promoting burn wound healing.
The invention has the following beneficial effects:
the invention provides a double-layer antibacterial dressing loaded with antibacterial molecules, and preparation and application thereof. The double-layer antibacterial dressing comprises: the fish skin gelatin nano fiber layer and the acellular fish skin matrix layer are loaded with antibacterial molecules, wherein the thickness of the fish skin gelatin nano fiber layer is 0.05-2mm, the diameter of the fish skin gelatin nano fiber is 100-600nm, and the thickness of the acellular fish skin matrix layer is 0.01-1mm. From the structure and the components, on one hand, the acellular fish skin has a bionic structure similar to that of a pig and a cowhide, also contains more abundant fat and omega-3 fatty acid, can obviously promote the generation of new blood vessels in a wound area, has an obvious promotion effect on wound repair, particularly repair of a difficult wound surface, can more effectively induce wound repair, has low immunogenicity of an acellular fish skin matrix, has good safety in clinical use, has a far lower risk of spreading viruses with human and livestock than pig and cowhide derived from land mammals, and cannot cause the body autoimmune reaction, on the other hand, after being combined with the fish skin gelatin nanofiber layer containing the antibacterial peptide, the outer fish skin gelatin nanofiber layer can provide a barrier effect for the inner acellular fish skin matrix layer, reduces the influence of the external environment, effectively protects the acellular fish skin, better plays a tissue repair role, and can further improve the antibacterial activity of a double-layer dressing by adding the antibacterial peptide, so that the acellular fish skin gelatin nanofiber layer is expected to be widely applied to the field of burn wound healing.
Drawings
The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
FIG. 1 shows a photograph of a physical embodiment of a two-layer dressing prepared in example 1 of the present invention.
FIG. 2 shows a scanning electron micrograph of the acellular fish skin matrix layer in the two-layer dressing prepared in example 1 of the present invention.
Fig. 3 shows a scanning electron micrograph of the antibacterial molecule-loaded fish skin gelatin nanofiber layer in the double-layered dressing prepared in example 1 of the present invention.
Fig. 4 shows a scanning electron microscope image of the cross section of the double-layered dressing prepared in example 1 of the present invention.
Fig. 5 shows a cell viability statistical map of the samples prepared in example 1, example 2 and comparative example 1.
Detailed Description
In order to more clearly illustrate the present invention, the present invention is further described below with reference to preferred embodiments and the accompanying drawings. Similar parts in the figures are denoted by the same reference numerals. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and is not to be taken as limiting the scope of the invention.
Example 1
(1) Removing scales, fish meat and inner fascia from fresh fish skin, soaking and kneading with saturated sodium chloride aqueous solution, repeatedly washing with ultrapure water, and draining;
(2) Soaking the fish skin in the step (1) with 2% formic acid solution for several times to perform degreasing and deproteinization treatment, cleaning with 4% sodium hydroxide solution, then repeatedly cleaning with ultrapure water, and draining;
(3) Freezing and thawing the fish skin obtained in the step (2) for a plurality of times at-80 ℃, then adopting 0.4% sodium dodecyl sulfate solution for cell removal treatment, then repeatedly cleaning with ultrapure water, and draining water;
(4) And (4) fixing the fish skin in the step (3) by using a clamp, and then freezing and drying at the temperature of minus 20 ℃ to obtain the acellular fish skin matrix with complete three-dimensional structure and smooth appearance.
(5) Adding fish skin enzymolysis gelatin into ultrapure water, and preparing into 10% fish skin gelatin aqueous solution at 50 ℃;
(6) Adding 0.8% of beta-defensin into the fish skin gelatin aqueous solution in the step (5), and dissolving by ultrasonic to prepare an electrostatic spinning precursor solution;
(7) Fixing the fish skin matrix obtained in the step (4) on a receiver of spiral electrostatic spinning equipment, adding the spinning precursor solution obtained in the step (6) into a spinning groove, spinning at the voltage of 20kV to obtain a layer of acellular fish skin matrix, wherein one layer is a double-layer antibacterial dressing precursor of a fish skin gelatin nanofiber layer, and then cutting the acellular fish skin matrix into a specification of 5cm multiplied by 5 cm;
(8) Hot-pressing and attaching the double-layer antibacterial dressing precursor in the step (7) at 50 ℃;
(9) Crosslinking the material in the step (8) by using a 1% glutaraldehyde solution for 24 hours, and then placing the material in a 35 ℃ forced air drying oven for drying for 48 hours;
(10) And (5) sterilizing the material in the step (9) by electron beam irradiation, wherein the irradiation dose is 10kGy, and finally obtaining the double-layer antibacterial dressing after vacuum packaging.
Example 2
(1) Removing scales, fish meat and inner fascia from fresh fish skin, soaking and kneading with saturated sodium chloride aqueous solution, repeatedly washing with ultrapure water, and draining;
(2) Soaking the fish skin in the step (1) with 2% formic acid solution for several times to perform degreasing and deproteinization treatment, cleaning with 4% sodium hydroxide solution, then repeatedly cleaning with ultrapure water, and draining;
(3) Freezing and thawing the fish skin obtained in the step (2) for a plurality of times at-80 ℃, then adopting a 0.4% sodium dodecyl sulfate solution for cell removal treatment, then repeatedly cleaning with ultrapure water, and draining water;
(4) And (4) fixing the fish skin in the step (3) by using a clamp, and then freezing and drying at the temperature of minus 20 ℃ to obtain the acellular fish skin matrix with complete three-dimensional structure and smooth appearance.
(5) Adding fish skin enzymolysis gelatin into ultrapure water, and preparing into 10% fish skin gelatin aqueous solution at 50 ℃;
(6) Adding 0.8% of bovine lactoferrin antibacterial peptide into the fish skin gelatin aqueous solution in the step (5), and dissolving by ultrasonic to prepare gelatin electrostatic spinning precursor solution;
(7) Fixing the fish skin matrix obtained in the step (4) on a receiver of spiral electrostatic spinning equipment, adding the spinning precursor solution obtained in the step (6) into a spinning groove, spinning at the voltage of 20kV to obtain a layer of acellular fish skin matrix, wherein one layer is a double-layer antibacterial dressing precursor of a fish skin gelatin nanofiber layer, and then cutting the acellular fish skin matrix into a specification of 5cm multiplied by 5 cm;
(8) Hot-pressing and attaching the double-layer antibacterial dressing precursor in the step (7) at 50 ℃;
(9) Crosslinking the material in the step (8) by using a 1% glutaraldehyde solution for 24 hours, and then drying the material in a 35 ℃ forced air drying oven for 48 hours;
(10) Sterilizing the material in the step (9) by electron beam irradiation with the irradiation dose of 10kGy, and carrying out vacuum packaging;
comparative example 1
(1) Removing scales, fish meat and inner fascia from fresh fish skin, soaking and kneading with saturated sodium chloride aqueous solution, repeatedly washing with ultrapure water, and draining;
(2) Soaking the fish skin in the step (1) with 2% formic acid solution for several times to perform degreasing and deproteinization treatment, cleaning with 4% sodium hydroxide solution, then repeatedly cleaning with ultrapure water, and draining;
(3) Freezing and thawing the fish skin obtained in the step (2) for a plurality of times at-80 ℃, then adopting a 0.4% sodium dodecyl sulfate solution for cell removal treatment, then repeatedly cleaning with ultrapure water, and draining water;
(4) Fixing the fish skin in the step (3) by using a clamp, freezing and drying at-20 ℃ to obtain a decellularized fish skin matrix with complete three-dimensional structure and smooth appearance, and then cutting into the specification of 5cm multiplied by 5 cm;
(6) Crosslinking the material in the step (5) by using a 1% glutaraldehyde solution for 24 hours, and then drying the material in a 35 ℃ forced air drying oven for 48 hours;
(7) Sterilizing the material in the step (6) by electron beam irradiation with the irradiation dose of 10kGy, and carrying out vacuum packaging;
experimental example 1
The experimental example is a bacteriostatic experiment test, and the test method comprises the following steps: e.coli and S.aureus were aseptically inoculated in a tryptic soy medium and cultured in a shaker at 37 ℃ for 16h. Adding 80 μm of the inoculum into 4mL of pancreatin soybean culture medium, and culturing for 2-4h. Then 106CFU/ml PBS bacterial suspension is obtained by a gradient dispersion method, and then the samples prepared in example 1, example 2 and comparative example 1 are added into the PBS bacterial suspension prepared in the above way, and the concentration is 0.4cm 2 mL, reaction at 37 ℃ for 1h. Then, 10. Mu.L each of the solutions was diluted 104-fold in sterile PBS. Subsequently, 100. Mu.L of the diluted PBS suspension was spread onto agar plates, all the agar plates were incubated at 37 ℃ for 16h, and finally, the number of suitable colonies formed was counted, and 3 parallel experiments were performed per group, and the average value was taken, and the test results are shown in Table 1 below. As can be seen from table 1, the samples prepared in examples 1 and 2 have excellent bacteriostatic properties against e.coli and s.aureus.
Table 1 comparative table of test data of bacteriostatic experiments of example 1, example 2 and comparative example 1
Experimental example 2
The experimental example is a cytotoxicity experimental test, and the test method is as follows: leaching liquor is prepared according to the national standard GB/T16886.12-2005, and the samples prepared in example 1, example 2 and comparative example 1 are respectively added into a cell culture medium, and the leaching ratio is 3cm 2 mL, culturing at 37 deg.C for 24h, refrigerating the obtained extract at 4 deg.C, adding the extract to a 96-well plate, inoculating 2000 cells per well, adding 10% FBS per well, at 37 deg.CBy 5% of CO 2 Culturing for 7 days, using the Incucyte as control group in the area of the well plate without adding leaching liquor TM The Zoom system observes the effect of the extract on human embryonic lung fibroblasts (MRC-5) in real time, and the test results are shown in FIG. 5. As can be seen from fig. 5, the relative cell survival rates of the samples prepared in comparative example 1 and example 1, example 2 were comparable to the control group, indicating that the prepared samples were not cytotoxic.
It should be understood that the above-described embodiments of the present invention are examples for clearly illustrating the invention, and are not to be construed as limiting the embodiments of the present invention, and it will be obvious to those skilled in the art that various changes and modifications can be made on the basis of the above description, and it is not intended to exhaust all embodiments, and obvious changes and modifications can be made on the basis of the technical solutions of the present invention.
Claims (10)
1. A bi-layer antimicrobial dressing loaded with antimicrobial molecules, the bi-layer antimicrobial dressing comprising: a fish skin gelatin nanofiber layer and a decellularized fish skin matrix layer which are loaded with antibacterial molecules; wherein the thickness of the fish skin gelatin nanofiber layer is 0.05-2mm, the diameter of the fish skin gelatin nanofiber is 100-600nm, and the thickness of the acellular fish skin matrix layer is 0.01-1mm.
2. The bi-layer antimicrobial dressing of claim 1, wherein the layer of antibacterial molecule-loaded fish skin gelatin nanofibers is formed on the layer of decellularized fish skin matrix by an electrospinning technique.
3. The bi-layer antimicrobial dressing of claim 1, wherein the antimicrobial molecule comprises an antimicrobial peptide; preferably, the antimicrobial peptide includes one or more of insect antimicrobial peptide, mammal antimicrobial peptide, amphibian antimicrobial peptide, fish antimicrobial peptide, mollusk antimicrobial peptide, crustacean antimicrobial peptide, plant antimicrobial peptide or bacterial antimicrobial peptide.
4. A method for preparing a bi-layer antimicrobial dressing according to any one of claims 1 to 3, comprising the steps of:
dissolving fish skin enzymolysis gelatin in water, preparing an electrostatic spinning precursor solution with antibacterial molecules, performing electrostatic spinning on the acellular fish skin matrix layer by using an electrostatic spinning technology to form a fish skin gelatin nanofiber layer, and performing hot-pressing bonding, crosslinking and drying to obtain the double-layer antibacterial dressing.
5. The method according to claim 4, wherein the concentration of the fish skin enzymatic gelatin in water is 5 to 15%; in the electrostatic spinning precursor solution, the concentration of antibacterial molecules is 0.5-1%; the temperature for preparing the electrostatic spinning precursor solution is 45-60 ℃.
6. The method according to claim 4, wherein the voltage of the electrospinning is 15 to 25kV, and the spraying distance of the needle of the syringe for electrospinning is 7 to 25cm.
7. The preparation method according to claim 4, wherein the hot press bonding temperature is 40-60 ℃; the hot-pressing laminating time is 0.1-30 s;
preferably, the cross-linking agent selected for cross-linking is one or more of glutaraldehyde, genipin, formaldehyde, epichlorohydrin and sodium trimetaphosphate; preferably, the concentration of the cross-linking agent is 1-5%; preferably, the crosslinking time is 24-48 h;
preferably, the drying temperature is 30-50 ℃, and the drying time is 24-48 h.
8. The method according to claim 4, further comprising sterilizing the dried product; preferably, the sterilization is one of high-pressure steam sterilization, co60 sterilization, electron beam irradiation sterilization or ethylene oxide sterilization; preferably, the sterilization is performed by electron beam irradiation; preferably, the irradiation dose of the electron beam irradiation sterilization is 10-20 KGy, and the irradiation time is 0.1-7 s.
9. The method of claim 4, wherein the layer of decellularized fish skin matrix is prepared by:
removing scales, fish flesh and inner fascia from fresh fish skin, soaking in saturated sodium chloride aqueous solution, washing with alkali liquor after acid treatment, repeatedly freezing and thawing at-60 ℃, taking out, then carrying out decellularization with a surfactant, washing with alkali liquor, and carrying out freeze drying to obtain a decellularized fish skin matrix layer;
preferably, the acid solution for acid treatment comprises one or more of an acetic acid solution, a citric acid solution, a phosphoric acid solution or a formic acid solution; preferably, the concentration of the acid liquor is 1-3%;
preferably, the alkali solution comprises one or more of sodium hydroxide, potassium hydroxide, calcium hydroxide or ammonia water; preferably, the concentration of the alkali liquor is 3-5%;
preferably, the surfactant comprises one or more of sodium dodecyl sulphate, tween 80, sodium deoxycholate or Triton X-100; preferably, the concentration of the surfactant is 0.3 to 0.5%.
10. Use of a bi-layer antimicrobial dressing according to any one of claims 1 to 3 or a bi-layer antimicrobial dressing prepared by the method of manufacture according to any one of claims 4 to 9 in the manufacture of a product for promoting healing of a burn wound.
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