CN117959499A - Bionic moist double-layer artificial skin based on silk and collagen and preparation method thereof - Google Patents
Bionic moist double-layer artificial skin based on silk and collagen and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a bionic moist double-layer artificial skin based on silk and collagen and a preparation method thereof, the bionic moist double-layer artificial skin has a double-layer structure, which respectively simulates a epidermis layer and a dermis layer of natural skin, the two layers of structures are compounded through chemical crosslinking, the epidermis layer is composed of silk-derived silk fibroin films, the bionic moist double-layer artificial skin has compact and smooth morphology, the tensile modulus is about 40MPa, and the stretching resistance and the protection effect can be provided; the dermis layer is composed of collagen hydrogel, has loose and porous morphology, and can provide space for cell adhesion and growth. The collagen hydrogel of the dermis layer has the effects of promoting cell adhesion and proliferation, and can enable the dermis layer to be regenerated rapidly. Therefore, the double-layer artificial skin provided by the invention can be used for treating skin defects caused by burn, mechanical injury and tumor excision, can also be used for treating difficult-to-heal skin ulcers caused by type II diabetes, renal dysfunction and the like, and can also be used as a three-dimensional bracket for skin cell culture.
Description
Technical Field
The invention relates to bionic wet double-layer artificial skin and a preparation method thereof, in particular to bionic wet double-layer artificial skin based on silk and collagen and a preparation method thereof.
Background
The skin is the largest organ of human body, and plays a very important role in aspects of immune protection, sensory conduction, body temperature regulation, human body beauty and the like. Wounds and tumor excision often result in large areas of full-thickness skin defects. At present, the clinic treatment of large-area full-layer skin tissue defects mainly adopts autologous skin transplantation (skin flap repair), allogenic skin transplantation and xenogenic skin transplantation. The traditional treatment methods have the risks of donor shortage, immune rejection, virus transmission and the like.
The advent of tissue engineering technology has brought new technological means for the regeneration of large-size skin defects. Traditional scaffolds for skin tissue engineering consist of biological materials and bioactive factors (e.g., cells and growth factors, etc.). The addition of cells or growth factors, while promoting skin healing, also makes tissue engineering skin scaffolds more expensive and more complex to control quality. Therefore, development of tissue engineering skin scaffolds containing only biological materials to achieve efficient skin regeneration has wider transformation prospects and clinical significance.
The invention patent CN101716375B discloses a preparation method of double-layer artificial skin, wherein the epidermal layer is a film prepared from silk fibroin and chitosan, and the dermal layer is a porous sponge prepared from collagen and sodium hyaluronate. The artificial skin is not chemically crosslinked, and is a dry scaffold with poor mechanical strength (tensile modulus 7.0 MPa). The invention aims to construct a bionic moist double-layer artificial skin, wherein an epidermis layer consists of silk fibroin from mulberry silk, a dermis part consists of collagen hydrogel, and the epidermis layer and the dermis layer are integrated through chemical crosslinking. The artificial skin has a tensile modulus of about 40.0MPa, and can promote cell adhesion and proliferation, and can be used for promoting skin tissue regeneration.
Disclosure of Invention
The invention aims to provide a double-layer artificial skin based on silk and collagen, a preparation method and application thereof, wherein the artificial skin simulates the structure, extracellular matrix components and functions of natural skin, has stretching resistance, can promote the adhesion and proliferation of human fibroblasts, and can be applied to the repair and regeneration of full-layer skin defects.
The technical scheme adopted by the invention is as follows:
The invention discloses a bionic moist double-layer artificial skin based on silk and collagen, which comprises an epidermis layer and a dermis layer, wherein the epidermis layer is a silk fibroin wet film with a tensile modulus of more than 40MPa, the dermis layer is collagen hydrogel, and the epidermis layer and the dermis layer are integrated through chemical crosslinking to form the double-layer artificial skin.
The invention also discloses a preparation method of the bionic moist double-layer artificial skin based on silk and collagen, which comprises the following steps:
1) Extracting silk fibroin from silk, and preparing a silk fibroin wet film epidermis;
2) Pouring the collagen solution above the silk fibroin wet film epidermis, and performing chemical crosslinking to obtain a double-layer bracket;
3) Washing the chemically crosslinked double-layer bracket to obtain the bionic wet double-layer artificial skin.
As a further improvement, the preparation method of the epidermal silk fibroin wet film comprises the following preparation steps:
1) Degumming: placing silkworm silk into sodium carbonate aqueous solution, boiling for degumming, taking out, cleaning with purified water to obtain silk fibroin, drying and preserving at room temperature;
2) Dissolving: dissolving the dried silk fibroin obtained in the step 1) in a lithium bromide solution, and heating in a water bath until the silk fibroin is completely dissolved to obtain a silk fibroin/lithium bromide solution;
3) And (3) dialysis: loading the silk fibroin/lithium bromide solution obtained in the step 2) into a regenerated cellulose dialysis bag, and putting into purified water for dialysis to remove lithium bromide, thereby obtaining a purified water solution of silk fibroin;
4) Freezing the purified water solution of the silk fibroin overnight, transferring to a freeze dryer, and freeze-drying to obtain freeze-dried regenerated silk fibroin sponge;
5) Dissolving the regenerated silk fibroin sponge obtained in the step 4) by deionized water to obtain a regenerated silk fibroin solution;
6) Adding the silk fibroin solution obtained in the step 5) into a mould, and then thoroughly drying at 0-65 ℃ to obtain a dried silk fibroin film;
7) Soaking in ethanol water solution for 2-6 hr, eliminating ethanol, adding deionized water to eliminate dried silk fibroin film, setting at 25-75 deg.c for 0.5-6 hr, and adding physiological saline to obtain the wet crosslinked silk fibroin epidermis rack.
As a further improvement, the silk fibroin wet film is prepared from silk fibroin extracted from mulberry silkworm silk.
As a further improvement, the die in the step 6) is a stainless steel dish or a polytetrafluoroethylene polymer die.
As a further improvement, the regenerated cellulose dialysis bag in step 3) according to the invention has a molecular weight cut-off of 5000-14000 daltons.
As a further improvement, the preparation method of the invention comprises the following steps of:
1) Adding a collagen solution onto the silk fibroin wet film skin layer;
2) Adding a chemical cross-linking agent into the mould for cross-linking to obtain a double-layer bracket with a lower layer of silk fibroin wet film and an upper layer of collagen hydrogel;
3) And (3) cleaning the double-layer scaffold obtained in the step (2) by using phosphate buffer solution to obtain wet double-layer artificial skin.
As a further improvement, the collagen in the collagen solution in step 1) is derived from animal tissue or from microbial fermentation or mammalian cell expression, wherein the collagen is any one of type i collagen, type iii collagen, type xvii collagen and type V ii collagen, the animal tissue is pigskin or bovine achilles tendon or fish skin or fish scale, and the microbial fermentation and mammalian cell expression is recombinant collagen.
As a further improvement, the chemical crosslinking agent in the step 2) is any one or a combination of any several of carbodiimide, diamine, epoxy compound, hydroxysuccinimide, glutaraldehyde and genipin.
As a further improvement, when the chemical cross-linking agent is carbodiimide and hydroxysuccinimide, the molar ratio of the carbodiimide to the hydroxysuccinimide is 3:1-8:1.
Compared with the prior art, the invention has the following innovation and beneficial effects:
1. The invention constructs the double-layer moist artificial skin which simulates human skin from three dimensions of spatial structure, material substance components and biological functions by using two kinds of natural materials with wide sources, and provides a new strategy for repairing and regenerating skin tissues.
2. The double-layer scaffold with the structure simulating natural skin is prepared, wherein the silk fibroin epidermis layer has compact structure and hydrophobic protection function, the collagen hydrogel layer simulates natural dermis, the structure is loose and porous, and a proper microenvironment can be provided for cell adhesion and growth. (corresponding to FIG. 3)
3. The invention utilizes chemical reaction to crosslink the silk fibroin wet film epidermis and the collagen hydrogel dermis, so that the epidermis and the dermis are connected through chemical bonds, and the integration is good.
4. The double-layer artificial skin stent is prepared based on silk and collagen, and the process is simple, so that the method is beneficial to mass production operation.
5. The polymer materials adopted in the preparation of the bionic wet double-layer artificial skin are degradable natural biological materials, have good biocompatibility, can be degraded along with skin tissue regeneration, and do not need to be peeled off and removed after being used.
6. The bionic moist double-layer artificial skin prepared by the invention has excellent mechanical properties (the tensile modulus is up to 40.0 MPa), can resist stretching, and has small limitation on the activity of an affected part. (corresponding FIGS. 4 and 5)
7. The dermis layer is collagen hydrogel, simulates the composition of natural skin dermis extracellular matrix, carries out substance component bionic, promotes cell adhesion and proliferation, and can accelerate tissue healing. (corresponding to FIGS. 6, 7, 8, 9)
8. The invention simultaneously defines the physicochemical properties of the bionic moist double-layer artificial skin based on silk and collagen, verifies the functions of the bracket by utilizing in-vitro cell experiments, and provides a basis for clinical application of the artificial skin.
Drawings
FIG. 1 is a schematic illustration of a bionic moist bilayer artificial skin based on silk and collagen;
FIG. 2 is an external view of a silk fibroin wet film skin and a bionic moist bilayer artificial skin (left SF: silk fibroin wet film skin; right SF/Col: bionic moist bilayer artificial skin);
FIG. 3 is a scanning electron microscope topography of a silk fibroin wet film epidermis and a biomimetic wetted bilayer artificial skin;
FIG. 4 is a graph of stress-strain for tensile testing of biomimetic wetted bilayer artificial skin with varying mass content epidermis;
FIG. 5 is a graph of stress-strain for tensile testing of a biomimetic wetted bilayer artificial skin with heat treated epidermis at different temperatures;
FIG. 6 is a graph showing the statistical results of the MTT assay for detecting the effect of silk fibroin wet film epidermis and biomimetic wet bilayer artificial skin scaffold on human fibroblast proliferation activity;
FIG. 7 is a graph showing the statistical results of the MTT assay for detecting the effect of silk fibroin wet film epidermis and biomimetic wet bilayer artificial skin scaffold on L929 cell proliferation activity;
FIG. 8 is a graph showing the statistical results of the effects of MTT assay on C3H cell proliferation activity of silk fibroin wet thin film epidermis and biomimetic wetted bilayer artificial skin scaffold;
FIG. 9 is a graph of statistical results of the effect of silk fibroin wet film epidermis and biomimetic wetted bilayer artificial skin scaffold on human fibroblast, L929 cells and C3H cell adhesion.
Detailed Description
The invention aims to provide a bionic moist double-layer artificial skin based on silk and collagen, which comprises an epidermis layer and a dermis layer, wherein the epidermis layer is a silk fibroin wet film with a tensile modulus of more than 40MPa, the dermis layer is collagen hydrogel, and the epidermis layer and the dermis layer are integrated through chemical crosslinking to form the double-layer artificial skin.
The artificial skin is prepared as follows:
(1) Taking silkworm silk as a raw material, degumming, washing, drying, dissolving and dialyzing to obtain trapped liquid, and freeze-drying the trapped liquid to obtain regenerated silk fibroin sponge; the regenerated silk fibroin sponge in the step (1) is a freeze-dried sponge, and is loose and porous. The preparation of the regenerated silk fibroin sponge is as follows: a) Degumming: boiling silkworm silk in sodium carbonate water solution for degumming, then washing with purified water to wash sericin, and drying and preserving the remaining sericin; b) Dissolving: dissolving the silk fibroin obtained in the step a) in a lithium bromide solution, and heating in a water bath until the silk fibroin is completely dissolved; c) And (3) dialysis: loading the silk fibroin/lithium bromide solution obtained in the step b) into a regenerated cellulose dialysis bag, and putting the regenerated cellulose dialysis bag into purified water for dialysis to remove lithium bromide, thereby obtaining a purified water solution of silk fibroin; c) The purified aqueous solution of silk fibroin is frozen overnight, then transferred to a freeze dryer, and freeze-dried regenerated silk fibroin sponge is obtained after freeze drying, and can be used for preparing silk fibroin solution.
(2) Dissolving regenerated silk fibroin sponge by deionized water to obtain regenerated silk fibroin solution;
(3) Adding 10-50 mL of silk fibroin solution into a mould, then placing the mould at 0-65 ℃ for thorough drying to obtain a dried silk fibroin film, then soaking the silk fibroin film in ethanol water solution for 2-6 hours, then adding deionized water to soak the silk fibroin film, placing the mould at 25-75 ℃ for 0.5-6 hours, and then adding normal saline to obtain the moist cross-linked silk fibroin epidermal scaffold. The die is a stainless steel or polytetrafluoroethylene polymer die;
(4) Preparing a collagen solution; collagen may be derived from animal tissue (porcine or bovine Achilles tendon or fish skin or fish scale) or recombinant collagen produced by microbial fermentation or mammalian cell expression; collagen may be derived from animal tissue, including but not limited to bovine Achilles tendon, pig skin, fish scales, fish skin, etc., may be derived from microbial fermentation, including but not limited to E.coli, pichia pastoris, and may be derived from animal cells, including but not limited to human fibroblasts, chinese hamster ovary cells, etc.
(5) And adding the collagen solution to the wet silk fibroin epidermis still in the mould, adding a chemical cross-linking agent for cross-linking, and then cleaning by using a phosphate buffer solution to obtain the bionic wet double-layer artificial skin scaffold.
The technical solutions of the present invention will be clearly and completely elucidated below in conjunction with the drawings and specific embodiments, and it will be apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1 construction of bionic moist bilayer Artificial skin based on Silk and collagen
(1) Preparation of silk fibroin solution: a) Degumming: putting 20g of silkworm silk into 2L of 0.05M sodium carbonate aqueous solution, boiling for 30 minutes at 100 ℃, taking out, washing with purified water for 5 times, repeating the process for 2 times, removing sericin, remaining silk fibroin, and drying the silk fibroin at 60 ℃; the dosage of the 0.05M sodium carbonate solution is 100mL/g based on the mass of the silkworm silk; b) Dissolving: 10g of dry silk fibroin is dissolved in 50mL of 9.3M lithium bromide solution, and water bath is carried out at 60 ℃ for 1h until the silk fibroin is completely dissolved; the amount of the 9.3M lithium bromide solution is 5mL/g based on the mass of the silk fibroin; c) And (3) dialysis: loading the silk fibroin/lithium bromide solution obtained in the step b) into a regenerated cellulose dialysis bag (with a molecular weight cut-off of 8000 daltons) for dialysis, and dialyzing with purified water with 10 times of the volume of the silk fibroin/lithium bromide solution for three days to remove lithium bromide, thereby obtaining a purified water solution of the silk fibroin; c) And transferring the purified water solution of the silk fibroin to a freeze dryer for 4-5 days after freezing at the temperature of-80 ℃ for overnight, and obtaining the freeze-dried regenerated silk fibroin sponge. The lyophilized regenerated silk fibroin sponge was dissolved with deionized water to prepare 40mL of 200mg/mL silk fibroin solution.
(2) Preparation of silk fibroin-based artificial epidermis: pouring 40mL of the silk fibroin solution prepared in the step (1) into a die, wherein the die is a stainless steel dish with square bottom surface (20 cm multiplied by 20 cm) and 1cm high; then drying for 1-2 days at room temperature, pouring 200mL of 90% ethanol for soaking for 2 hours, removing 90% ethanol, adding deionized water, treating at 65 ℃ for 1 hour, and finally soaking in normal saline to obtain the wet silk fibroin epidermis.
(3) Compounding collagen-based artificial dermis with silk fibroin epidermis: the principle of which is shown in figure 1. Firstly, preparing a 10mg/mL collagen solution from the dried collagen powder by using 0.5M acetic acid buffer with pH of 2.5; 40mL of the collagen composite solution was added to the silk fibroin epidermis still in the mold, then N-hydroxysuccinimide (NHS) solution was added, and the pH was adjusted to 5.5 with MES as buffer, then 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) solution was added and mixed well, EDC: NHS molar ratio is 5:1, shaking table reaction is carried out for 24 hours for crosslinking, and finally PBS buffer solution is used for cleaning, so that wet double-layer artificial skin is obtained.
(4) And (3) sterilization: and carrying out irradiation treatment for more than 30 minutes by adopting 60 Co gamma rays or electron beams to obtain the silk/collagen double-layer artificial skin scaffold.
Fig. 1 shows a schematic diagram of the principle of preparing a biomimetic moist bilayer artificial skin based on silk and collagen. Figures 2 and 3 show the results of digital photographs and scanning electron microscope characterization of silk fibroin epidermal Scaffolds (SF) and silk/collagen bilayer artificial skin, respectively. As can be seen from the digital photographs, a transparent collagen hydrogel layer (Col) is covered on top of the chemically crosslinked silk fibroin epidermis (SF) scaffold. The scanning electron microscope characterization results further show that the SF skin layer is compact and flat, the Col dermis layer is visible in collagen fibers and void structures, and the interface of the SF skin layer and the Col dermis layer can be observed from the section under high magnification. Thus, the results demonstrate that the SF epidermis layer and Col dermis layer achieve cross-linking integration, i.e. successful construction of silk/collagen bilayer artificial skin.
Example 2 exploration of the Effect of silk fibroin content in epidermis on the tensile modulus of bionic moist bilayer bionic artificial skin
(1) Preparation of silk fibroin-based artificial epidermis with different content: first, a silk fibroin solution was prepared by the same method as in the step (1) of example 1, and 20mL and 40mL of the silk fibroin solution were poured into two stainless steel dish molds each having a square bottom surface (20 cm. Times.20 cm) and a height of 1 cm; then drying for 1-2 days at room temperature, pouring 200mL of 90% ethanol for soaking for 2 hours, removing 90% ethanol, adding deionized water, treating at 65 ℃ for 1 hour, and finally soaking in normal saline to obtain the two-mass-content moist silk fibroin epidermis.
(2) Compounding collagen-based artificial dermis with silk fibroin epidermis: the process is the same as in step (3) of example 1: preparing collagen powder into 10mg/mL collagen solution; two 40mL collagen solutions were added to two epidermis (1) with different silk fibroin content, then NHS solution was added to each, MES was used as buffer to adjust pH to 5.5, EDC solution was added and mixed well: NHS molar ratio is 5:1, shaking table reaction is carried out for 24 hours for crosslinking, and finally PBS buffer solution is used for cleaning, so that wet double-layer artificial skin is obtained.
(3) And (3) sterilization: same as in example 1 (4)
(4) Mechanical testing: the obtained double-layer artificial skin bracket is cut into a rectangular film with the thickness of 10mm multiplied by 50mm, the elastic modulus is detected by using a universal testing machine, and the parameters are set to be 20mm in clamping distance and 20mm/min in stretching speed. The stress-strain curve results are shown in fig. 4, and the tensile modulus of the two artificial skins was calculated from the linear portion of the curve. When the epidermis is made from 20mL silk fibroin solution, the tensile modulus of the artificial skin is about 23.42MPa; when the epidermis is worth 40mL of silk fibroin solution, the stretched film amount of the artificial skin is about 41.87MPa. It can be seen that the tensile modulus of artificial skin increases with increasing silk fibroin content in the epidermis.
Example 3 exploration of the Effect of different temperature Heat treatments on the mechanical Properties of bionic moist bilayer bionic Artificial skin
(1) Preparation of silk fibroin-based artificial epidermis: two 40mL silk fibroin solutions were prepared by the same method as in step (1) of example 1, and poured into two molds having square bottom surfaces (20 cm. Times.20 cm) and a height of 1cm, respectively; then drying at room temperature for 1-2 days, pouring 200mL of 90% ethanol, soaking for 2 hours, removing 90% ethanol, adding deionized water, respectively treating at 37 ℃ and 65 ℃ for 1 hour, and soaking in physiological saline to obtain two types of moist silk fibroin epidermis.
(2) The method for compounding collagen-based artificial dermis and silk fibroin epidermis and the method for sterilizing the same as in step (3) and step (4) in example 1
(3) Mechanical property test: cutting the obtained bionic wet double-layer artificial skin into a rectangular film with the thickness of 10mm multiplied by 50mm, detecting the elastic modulus of the rectangular film by using a universal testing machine, and setting parameters to be 20mm of clamp distance and 20mm/min of stretching rate. The stress-strain curve results are shown in fig. 5, and the tensile modulus of the two artificial skins was calculated from the linear portion of the curve. The tensile modulus of the artificial skin obtained by heat treatment at two temperatures is about 40MPa, so that the tensile modulus of the artificial skin is not obviously influenced by the heat treatment temperature range adopted by the method.
EXAMPLE 4 cell proliferation
The silk/collagen bilayer artificial skin prepared in example 1 was denoted as SF/Col, and the silk fibroin wet film skins prepared in example 1 and example 2 were each denoted as SF.
SF/Col and SF are respectively leached at leaching concentration of 3cm 2/mL, leaching liquor is DMEM high sugar culture medium containing 10% fetal calf serum, leaching is carried out for 72h at constant temperature of 37 ℃ to obtain artificial skin leaching liquor, and the temperature is 4 ℃ for standby. Human fibroblasts, L929 cells and C3H cells with good growth state are digested, resuspended and counted by using pancreatin. mu.L of human fibroblasts, L929 cells and C 3 H cell suspensions were inoculated into 96-well plates at 1000 cells per well, respectively, and cultured overnight in a constant temperature incubator at 37 ℃. The medium was aspirated, 100. Mu.L SF/Col and SF prepared artificial skin extract was added to each well, the extract was changed every three days, and 5 duplicate wells were placed in each group. Control groups were supplemented with 100 μl of DMEM high sugar medium containing 10% fetal bovine serum, the medium was changed every three days, and 5 duplicate wells were placed in each group. 10. Mu.L of MTT dye was added to the medium for staining after 1,3 and 7 days, respectively, and the culture was continued for 4 hours. The original medium in the well plate was aspirated, 150. Mu. LDMSO of the solution was added, mixed well, and absorbance at 450nm was measured using an microplate reader.
The proliferation effects of silk/collagen double-layer artificial skin extract on human fibroblasts, L929 cells and C3H cells are shown in figures 6,7 and 8 respectively, and the results show that the double-layer scaffold SF/Col can promote the proliferation of the fibroblasts, and the proliferation effect is superior to that of a silk fibroin epidermis without collagen.
EXAMPLE 5 cell adhesion
Mu.L of human fibroblasts, L929 cells and C 3 H cells were seeded at a concentration of 5X 10 4 cells/mL into a blank 6-well plate, 3mL of cell suspension was added to each well, and incubated overnight in a 37℃incubator. The medium was aspirated and 3mLSF/Col extract, SF extract and 10% foetal calf serum DMEM high sugar medium were added to each well. The control group is cell culture medium, the blank group is cell culture medium without cells, and each group is provided with 3-5 compound holes. Incubation and culture were performed in a constant temperature incubator at 37℃for 24 hours. The culture medium is sucked and removed, PBS is used for washing for 2 to 3 times, 3mL of calcein working solution is added into each hole, incubation and culture are carried out for 20 to 30min, supernatant is removed, and PBS is added for washing cells. Fluorescent microscopy was used to photograph and count cells using ImageJ software.
Cell adhesion results of silk/collagen bilayer artificial skin as shown in fig. 9, sf/Col has good adhesion ability to human fibroblasts, L929 cells and C 3 H cells.
The foregoing is not intended to limit the invention, and it should be noted that variations, modifications, additions and substitutions are possible, without departing from the scope of the invention as disclosed in the accompanying claims.
Claims (10)
1. The bionic moist double-layer artificial skin based on silk and collagen is characterized by comprising an epidermis layer and a dermis layer, wherein the epidermis layer is a silk fibroin wet film with a tensile modulus of more than 40MPa, the dermis layer is collagen hydrogel, and the epidermis layer and the dermis layer are integrated through chemical crosslinking to form the double-layer artificial skin.
2. A preparation method of bionic moist double-layer artificial skin based on silk and collagen is characterized by comprising the following steps:
1) Extracting silk fibroin from silk, and preparing a silk fibroin wet film epidermis;
2) Pouring the collagen solution above the silk fibroin wet film epidermis, and performing chemical crosslinking to obtain a double-layer bracket;
3) Washing the chemically crosslinked double-layer bracket to obtain the bionic wet double-layer artificial skin.
3. The method for preparing the bionic moist bilayer artificial skin based on silk and collagen according to claim 2, wherein the preparing the epidermal silk fibroin wet film comprises the following steps:
1) Degumming: placing silkworm silk into sodium carbonate aqueous solution, boiling for degumming, taking out, cleaning with purified water to obtain silk fibroin, drying and preserving at room temperature;
2) Dissolving: dissolving the dried silk fibroin obtained in the step 1) in a lithium bromide solution, and heating in a water bath until the silk fibroin is completely dissolved to obtain a silk fibroin/lithium bromide solution;
3) And (3) dialysis: loading the silk fibroin/lithium bromide solution obtained in the step 2) into a regenerated cellulose dialysis bag, and putting into purified water for dialysis to remove lithium bromide, thereby obtaining a purified water solution of silk fibroin;
4) Freezing the purified water solution of the silk fibroin overnight, transferring to a freeze dryer, and freeze-drying to obtain freeze-dried regenerated silk fibroin sponge;
5) Dissolving the regenerated silk fibroin sponge obtained in the step 4) by deionized water to obtain a regenerated silk fibroin solution;
6) Adding the silk fibroin solution obtained in the step 5) into a mould, and then thoroughly drying at 0-65 ℃ to obtain a dried silk fibroin film;
7) Soaking in ethanol water solution for 2-6 hr, eliminating ethanol, adding deionized water to eliminate dried silk fibroin film, setting at 25-75 deg.c for 0.5-6 hr, and adding physiological saline to obtain the wet crosslinked silk fibroin epidermis rack.
4. The method for preparing a bionic moist bilayer artificial skin based on silk and collagen according to claim 3, wherein the silk fibroin is prepared from silk fibroin extracted from mulberry silkworm silk.
5. The method for preparing the bionic moist bilayer artificial skin based on silk and collagen according to claim 3, wherein the mold in the step 6) is a stainless steel dish or a polytetrafluoroethylene polymer mold.
6. The method for preparing a bionic moist bilayer artificial skin based on silk and collagen according to claim 3 or 4, wherein the regenerated cellulose dialysis bag in step 3) has a molecular weight cut-off of 5000-14000 daltons.
7. The method for preparing the bionic wet double-layer artificial skin based on silk and collagen according to claim 6, wherein the step of pouring the collagen solution over the silk fibroin wet film epidermis and performing chemical crosslinking to obtain the double-layer scaffold specifically comprises the following steps:
1) Adding a collagen solution onto the silk fibroin wet film skin layer;
2) Adding a chemical cross-linking agent into the mould for cross-linking to obtain a double-layer bracket with a lower layer of silk fibroin wet film and an upper layer of collagen hydrogel;
3) And (3) cleaning the double-layer scaffold obtained in the step (2) by using phosphate buffer solution to obtain the wet double-layer artificial skin scaffold.
8. The method for preparing the bionic moist bilayer artificial skin based on silk and collagen according to claim 7, wherein the collagen in the collagen solution in the step 1) is derived from animal tissue or from microbial fermentation or expression of mammalian cells, the collagen is any one of type i collagen, type iii collagen, type xii collagen and type V ii collagen, the animal tissue is pig skin or bovine achilles tendon or fish skin or fish scale, and the microbial fermentation and expression of mammalian cells is recombinant collagen.
9. The method for preparing the bionic moist bilayer artificial skin based on silk and collagen according to claim 7 or 8, wherein the chemical crosslinking agent in the step 2) is any one or a combination of any several of carbodiimide, diamine, epoxy compound, hydroxysuccinimide, glutaraldehyde and genipin.
10. The method for preparing the bionic moist bilayer artificial skin based on silk and collagen according to claim 9, wherein when the chemical cross-linking agent is carbodiimide and hydroxysuccinimide, the molar ratio is 3:1-8:1.
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