CN115887785B - Antibacterial artificial skin and preparation method thereof - Google Patents
Antibacterial artificial skin and preparation method thereof Download PDFInfo
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- CN115887785B CN115887785B CN202211652205.9A CN202211652205A CN115887785B CN 115887785 B CN115887785 B CN 115887785B CN 202211652205 A CN202211652205 A CN 202211652205A CN 115887785 B CN115887785 B CN 115887785B
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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
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- Materials For Medical Uses (AREA)
Abstract
The invention provides an antibacterial artificial skin and a preparation method thereof, and belongs to the technical field of artificial skin, wherein the antibacterial artificial skin comprises a hypodermis layer, a hypodermis upper layer, an absorbable epidermis layer and a non-absorbable epidermis layer which are sequentially arranged from bottom to top; the hypodermis layer is a spongy porous collagen layer; the dermis upper layer is a porous acellular matrix layer; the porous decellularized matrix layer at least comprises a porous decellularized matrix film adsorbed with active substances; the active substance comprises an epidermal growth factor and an alkaline fibroblast growth factor; the absorbable epidermis layer is a bacteriostatic decellularized matrix layer; the antibacterial decellularized base layer comprises a decellularized matrix film with a surface coated with nano mesoporous bioactive glass; the non-absorbable skin layer is a film layer. The artificial skin provided by the invention has continuous antibacterial activity, can prevent wound infection, can effectively promote wound healing of soft tissue injury, improves tissue regeneration and repair capability, and accelerates the reconstruction of dermis.
Description
Technical Field
The invention belongs to the technical field of artificial skin, and particularly relates to antibacterial artificial skin and a preparation method thereof.
Background
The skin is the largest organ of the human body, and the injury can be caused by trauma, burn, diabetic foot, tumor excision operation and the like; at present, a repair method for deeper or large-area skin defects clinically is autologous skin graft or skin flap transplantation, but the autologous skin graft or skin flap transplantation can cause donor area injury; the allogenic skin patch or skin flap transplantation has the problems of immune rejection, safety in use and the like.
The artificial skin can be used as a substitute for autologous skin transplantation, can be used for treating wounds with damaged dermis layers, chronic wounds or burn wounds, has the effects of promoting wound healing, reducing scar formation and the like, overcomes the defect of insufficient autologous skin transplantation resources of large-area burn patients, and gradually occupies the main stream of the market. However, artificial skin fails to establish blood supply with wound surface in early stage after transplantation, has poor antibacterial ability and high infection rate after transplantation, and infection can not only directly lead to transplantation failure, but also threaten patient life. At present, the common measures for preventing infection after artificial skin transplantation in clinic mainly comprise: the method comprises the steps of thoroughly cleaning a wound surface, immersing artificial skin in a disinfectant before implantation, and enhancing dressing change after implantation, so that antibiotics are used throughout the body; although the above measures can play a role in preventing infection after transplantation to a certain extent, the artificial skin cannot be endowed with antibacterial capability, and a large amount of medicines are easy to cause damage to viscera of a patient, so that the body of the patient is negatively influenced.
Disclosure of Invention
Aiming at one or more technical problems in the prior art, the invention provides the antibacterial artificial skin and the preparation method thereof.
The present invention provides in a first aspect an antimicrobial artificial skin comprising a sub-dermal layer, an upper dermal layer, an absorbable epidermal layer and a non-absorbable epidermal layer disposed sequentially from bottom to top;
the hypodermis layer is a spongy porous collagen layer;
the dermis upper layer is a porous acellular matrix layer; the porous decellularized matrix layer at least comprises a porous decellularized matrix film adsorbed with active substances; the active substance comprises an epidermal growth factor and an alkaline fibroblast growth factor;
the absorbable epidermis layer is a bacteriostatic decellularized matrix layer; the antibacterial acellular matrix layer comprises an acellular matrix film with a surface coated with nano mesoporous bioactive glass;
the non-absorbable skin layer is a film layer.
Preferably, the spongy porous collagen layer is obtained by freeze-drying a collagen solution;
the thickness of the spongy porous collagen layer is 0.5-5 mm; the pore diameter of the spongy porous collagen layer is 20-200 mu m; the spongy porous collagen layer has a porosity of greater than 90%.
Preferably, the thickness of the porous decellularized matrix membrane is 0.02-0.1 mm; the porous acellular matrix membrane is provided with a plurality of through holes along the thickness direction, the diameter of the through holes is 0.1-1 mm, and the density of the through holes is 9-900/cm 2 。
Preferably, the thickness of the acellular matrix membrane is 0.02-0.1 mm; the mass ratio of the nano mesoporous bioactive glass to the acellular matrix membrane is 1 (9-49).
Preferably, the particle size of the nano mesoporous bioactive glass is not more than 50 mu m, and the pore diameter is 5-20 nm.
Preferably, the non-absorbable skin layer is a silicone rubber film or a polyvinyl alcohol film; the thickness of the non-absorbable skin layer is 0.1-0.25 mm.
The present invention provides in a second aspect a method for preparing the antibacterial artificial skin according to the first aspect, the method comprising the steps of:
s1, cleaning fresh tissues, and then carrying out disinfection, degreasing and decellularization treatment to obtain a decellularized matrix membrane;
s2, freeze-drying and perforating the acellular matrix membrane, and then soaking the acellular matrix membrane in a solution containing active substances to obtain a porous acellular matrix membrane adsorbed with the active substances, namely an upper acellular matrix membrane of dermis;
s3, the nanometer mesoporous bioactive glass is spread on the surface of the acellular matrix film, so that the acellular matrix film with the surface coated with the nanometer mesoporous bioactive glass is obtained, and the acellular matrix film of the epidermis layer can be absorbed;
s4, sequentially paving the absorbable epidermis layer acellular matrix membrane, the dermis upper layer acellular matrix membrane and the collagen solution in a mould, and compositing a non-absorbable epidermis layer on the absorbable epidermis layer after freeze drying to obtain the antibacterial artificial skin.
Preferably, in the step S1, the sterilization is to soak the slices after the fresh tissues are washed in an ethanol solution of peracetic acid for 30-60 min;
the degreasing is to soak the sterilized tissue in sodium hydroxide solution for 30-60 min, and then clean the tissue with phosphate buffer;
the decellularized treatment is to soak the defatted tissue in trypsin solution for 1-2 h to obtain the decellularized matrix layer.
Preferably, the solution containing the active substance is obtained by mixing a collagen solution, an epidermal growth factor and an alkaline fibroblast growth factor;
in the solution containing the active substances, the mass fraction of collagen is 0.5-0.9%, and the concentration of the epidermal cell growth factor is 5ug/mL; the concentration of the basic fibroblast growth factor is 5ug/mL.
Preferably, the collagen solution is obtained by mixing type I collagen with acetic acid solution; the mass fraction of acetic acid in the acetic acid solution is 4-8%.
Compared with the prior art, the invention has at least the following beneficial effects:
the porous decellularized matrix layer obtained by adopting the tissue decellularized process on the dermis upper layer has a strong mechanical property similar to a natural dermis structure, is coated with active substances, and can accelerate the reconstruction of the dermis layer; the hypodermis layer is a spongy porous collagen layer, which is beneficial to hemostasis and fibroblast and capillary blood tube growth and is beneficial to reconstruction of the hypodermis layer; the absorbable epidermis is a tissue-free cell matrix layer treated by adopting nano mesoporous bioactive glass, can promote cell adhesion and proliferation, improve tissue regeneration and repair capability, has continuous antibacterial activity, and can play a role similar to epidermis stratum corneum in the later stage when achieving the purpose of preventing wound infection; the non-absorbable skin layer is a silicon rubber film or a polyvinyl alcohol film, and can play roles in preventing moisture loss, isolating, preventing bacteria from invading and the like.
The dermis upper layer and the dermis lower layer adopt materials with different sources and processes to control degradation and tissue reconstruction time, and the degradation and tissue reconstruction time can be controlled through different layering layers, so that different clinical requirements are met. The artificial skin provided by the invention has continuous antibacterial activity, can prevent wound infection, can effectively promote wound healing of soft tissue injury, improves tissue regeneration and repair capability, and accelerates the reconstruction of dermis.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic structural view of bacteriostatic artificial skin provided by the invention;
FIG. 2 is a flow chart of a preparation method of the antibacterial artificial skin provided by the invention;
in the figures, the 1-subdermal layer, the 2-subdermal layer, the 3-absorbable epidermis layer, the 4-non-absorbable epidermis layer.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the embodiments described below will be clearly and completely described in conjunction with the technical solutions of the embodiments of the present invention, and it is apparent that the described embodiments are some, but not all, embodiments of the present invention, and all other embodiments obtained by persons of ordinary skill in the art without making any inventive effort based on the embodiments of the present invention are within the scope of protection of the present invention.
As shown in fig. 1, the present invention provides, in a first aspect, an antimicrobial artificial skin comprising a sub-dermal layer 1, an upper dermal layer 2, an absorbable epidermal layer 3 and a non-absorbable epidermal layer 4 disposed in this order from bottom to top;
the hypodermis layer 1 is a spongy porous collagen layer;
the dermis upper layer 2 is a porous decellularized matrix layer; the porous decellularized matrix layer at least comprises a porous decellularized matrix film adsorbed with active substances; the active substance comprises Epidermal Growth Factor (EGF) and basic fibroblast growth factor (bFGF);
the absorbable epidermis layer 3 is a bacteriostatic decellularized matrix layer; the antibacterial acellular matrix layer comprises an acellular matrix film with a surface coated with nano mesoporous bioactive glass;
the non-absorbable skin layer 4 is a silicone rubber film or a polyvinyl alcohol film.
In the present invention, "upper" and "lower" are defined based on the positional relationship of the layers in fig. 1.
The porous decellularized matrix layer obtained by adopting the tissue decellularized process on the dermis upper layer has a strong mechanical property similar to a natural dermis structure, is coated with active substances, and can accelerate reconstruction of dermis; the hypodermis layer is a spongy porous collagen layer, which is beneficial to hemostasis and fibroblast and capillary blood tube growth and is beneficial to reconstruction of the hypodermis layer; the absorbable epidermis is a tissue-free cell matrix layer treated by adopting nano mesoporous bioactive glass, can promote cell adhesion and proliferation, improve tissue regeneration and repair capability, has continuous antibacterial activity, can promote healing of soft tissue injury wound surface while achieving the purpose of preventing wound surface infection, and can play a role similar to epidermis stratum corneum in the later stage; the non-absorbable skin layer is a silicon rubber film or a polyvinyl alcohol film, and can play roles in preventing moisture loss, isolating, preventing bacteria from invading and the like; the artificial skin of the present invention (the portion from which the non-absorbable skin layer is removed) has a tensile strength of not less than 5N/cm.
The dermis upper layer and the dermis lower layer adopt materials with different sources and processes to control degradation and tissue reconstruction time, and the degradation and tissue reconstruction time can be controlled through different layering layers, so that different clinical requirements, such as common skin defects or diabetic feet, bedsores and the like, are met. The non-absorbable skin layer was not sutured, but the absorbable skin layer was sutured; when the artificial skin is trimmed, a part of non-absorbable epidermis layer can be reserved for covering so as to avoid direct exposure of the part to be trimmed; after vascularization of the newly generated dermal tissue is completed, the non-absorbable epidermis layer can be removed.
According to some preferred embodiments, the spongy porous collagen layer is obtained by freeze-drying a collagen solution;
the thickness of the spongy porous collagen layer is 0.5 to 5mm (for example, may be 0.5mm, 1mm, 1.5mm, 2mm, 2.5mm, 3mm, 3.5mm, 4mm, 4.5mm or 5 mm); the porous collagen sponge layer has a pore size of 20 to 200 μm (e.g., may be 20 μm, 50 μm, 60 μm, 80 μm, 100 μm, 120 μm, 140 μm, 160m, 180 μm, or 200 μm); the spongy porous collagen layer has a porosity of greater than 90%.
The hypodermis layer is a spongy porous collagen layer, and the spongy porous collagen layer is a porous structure obtained by freeze drying of a collagen solution, and is suitable for cell adhesion and migration when the pore size of the hypodermis layer is 20-200 mu m and the porosity is more than 90%, thereby being beneficial to connective tissue growth, formation of new blood vessels and skin regeneration.
According to some preferred embodiments, the porous decellularized matrix film has a thickness of 0.02 to 0.1mm (e.g., can be 0.02mm, 0.03mm, 0.04mm, 0.05mm, 0.06mm, 0.07mm, 0.08mm, 0.09mm, or 0.1 mm); the porous decellularized matrix film is provided with a plurality of through holes in the thickness direction, the diameter of the through holes is 0.1-1 mm (for example, 0.1mm, 0.2mm, 0.4mm, 0.8mm or 1 mm), and the density of the through holes is 9-900 pieces/cm 2 (e.g., may be 9/cm) 2 50 pieces/cm 2 100 pieces/cm 2 150 pieces/cm 2 200 pieces/cm 2 250 pieces/cm 2 300 pieces/cm 2 350 pieces/cm 2 400 pieces/cm 2 450 pieces/cm 2 500 pieces/cm 2 550 pieces/cm 2 600 pieces/cm 2 650 pieces/cm 2 700 pieces/cm 2 750/cm 2 800 pieces/cm 2 850 pieces/cm 2 Or 900 pieces/cm 2 )。
It should be noted that, in the present invention, the density of the through holes is adjusted according to the aperture of the through holes, and the larger the aperture is, the smaller the density of the through holes is; for example, when the hole diameter of the through holes is 0.1mm, the density of the through holes is not more than 900 pieces/cm 2 The preparation method is finished; when the hole diameter of the through holes is 1mm, the density of the through holes is not more than 9 pieces/cm 2 And the method can be specifically adjusted according to actual requirements.
The dermis upper layer is at least one porous acellular matrix layer of a porous acellular matrix membrane adsorbed with active substances, and the porous acellular matrix membrane is obtained by perforating the acellular matrix membrane; the decellularized matrix membrane is obtained by a decellularized process, has a structure similar to that of natural dermis and has good mechanical properties; the active substances can heal the wound surface of soft tissue injury, and the dermis reconstruction is quickened; the number of layers of the porous acellular matrix membrane in the upper dermis is not particularly limited, and is designed and adjusted according to actual conditions.
According to some preferred embodiments, the decellularized matrix film has a thickness of 0.02 to 0.1mm (e.g., can be 0.02mm, 0.03mm, 0.04mm, 0.05mm, 0.06mm, 0.07mm, 0.08mm, 0.09mm, or 0.1 mm); the mass ratio of the nano mesoporous bioactive glass to the acellular matrix membrane is 1 (9-49) (e.g., can be 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, or 1:49).
The absorbable epidermis layer is a bacteriostatic acellular matrix layer of which the surface is coated with the acellular matrix film of the nano mesoporous bioactive glass, and the acellular matrix film is obtained by adopting an acellular process, is similar to a natural dermis structure, and has good mechanical properties; the nanometer mesoporous bioactive glass is bioactive glass prepared by a sol-gel process, can promote cell adhesion and proliferation, further improve tissue regeneration and repair capability, and can be used as a bioactive component for promoting angiogenesis; meanwhile, the nano mesoporous bioactive glass has continuous antibacterial activity, and can promote healing of soft tissue injury wound surfaces while achieving the purpose of preventing wound surface infection; if the nano mesoporous bioactive glass is not added, the prepared artificial skin has poor antibacterial effect.
According to some preferred embodiments, the nano-mesoporous bioactive glass has a particle size of no greater than 50 μm and a pore size of 5-20 nm (e.g., may be 5nm, 8nm, 10nm, 12nm, 15nm, 18nm, or 20 nm).
According to some preferred embodiments, the non-absorbable skin layer is a silicone rubber film or a polyvinyl alcohol film; the non-absorbable skin layer 4 has a thickness of 0.1 to 0.25mm (for example, may be 0.1mm, 0.12mm, 0.14mm, 0.16mm, 0.18mm, 0.2mm, 0.22mm, 0.24mm, or 0.25 mm).
As shown in fig. 2, the present invention provides, in a second aspect, a method for preparing the antibacterial artificial skin according to the first aspect, the method comprising the steps of:
s1, cleaning fresh tissues, and then carrying out disinfection, degreasing and decellularization treatment to obtain a decellularized matrix membrane;
s2, freeze-drying and perforating the acellular matrix membrane, and then soaking the acellular matrix membrane in a solution containing active substances to obtain a porous acellular matrix membrane adsorbed with the active substances, namely an upper acellular matrix membrane of dermis;
s3, the nanometer mesoporous bioactive glass is spread on the surface of the acellular matrix film, so that the acellular matrix film with the surface coated with the nanometer mesoporous bioactive glass is obtained, and the acellular matrix film of the epidermis layer can be absorbed;
s4, sequentially paving the absorbable epidermis layer acellular matrix membrane, the dermis upper layer acellular matrix membrane and the collagen solution in a mould, and compositing a non-absorbable epidermis layer on the absorbable epidermis layer after freeze drying to obtain the antibacterial artificial skin.
The freeze drying comprises a pre-freezing stage, a first sublimation stage and a second sublimation stage, and the target temperature and the target duration of each stage are as follows:
pre-freezing: freezing at-30deg.C for 30min without adding sample, and freezing at-30deg.C for 90min;
a first sublimation stage: vacuumizing, aerating at 100+ -10 Pa, freezing at-10deg.C for 60min, and freezing at 0deg.C for 120min;
a second sublimation stage: vacuumizing, aerating at 100+ -10 Pa, freezing at 10deg.C for 120min, freezing at 20deg.C for 120min, and freezing at 25deg.C for 120min; and (5) when the temperature of the front temperature box reaches 25 ℃, shutting down.
The collagen solution is prepared by adopting fresh tissues (small intestine, amniotic membrane, dura mater or bladder tissues) to carry out a decellularization process to obtain a decellularized matrix membrane, carrying out freeze-drying, punching on the surface of the obtained decellularized matrix membrane by using a punching tool, and then soaking in a solution containing active substances to obtain a porous decellularized matrix membrane adsorbed with the active substances, namely, a dermis upper layer decellularized matrix membrane; then, the surface of the acellular matrix membrane prepared by adopting fresh tissues (small pig intestine, amniotic membrane, dura mater or bladder tissues) to carry out a decellularization process is coated with nano mesoporous bioactive glass to obtain the acellular matrix membrane coated with the nano mesoporous bioactive glass on the surface, namely the acellular matrix membrane of the epidermis layer can be absorbed; and finally, sequentially paving the cell-free matrix membrane of the absorbable epidermis layer, the cell-free matrix membrane of the dermis upper layer and the collagen solution in a mould, and compositing the non-absorbable epidermis layer on the absorbable epidermis layer by adopting a medical adhesive coating after the whole freeze drying, so as to obtain the antibacterial artificial skin, wherein the medical adhesive coating is a silica gel adhesive layer, an acrylic acid coating or a polyurethane coating.
The invention can adjust the different layering layers of the upper dermis layer in the layering process, control the degradation and tissue reconstruction time and meet different clinical requirements.
According to some preferred embodiments, in step S1, the sterilization is to soak the slices after washing the fresh tissue in an ethanol solution of peracetic acid for 30-60 min (for example, 30min, 35min, 40min, 45min, 50min, 55min or 60 min); the concentration of the peracetic acid in the ethanol solution of peracetic acid according to the present invention was 0.2% by mass.
The degreasing is to soak the sterilized tissue in a sodium hydroxide solution for 30-60 min (for example, 30min, 35min, 40min, 45min, 50min, 55min or 60 min), and then wash the tissue with a phosphate buffer; the sodium hydroxide solution of the present invention was an aqueous sodium hydroxide solution having a concentration of 15mmol/L.
The decellularization treatment is to soak the defatted tissue in trypsin solution for 1-2 h (for example, 1h, 1.2h, 1.4h, 1.6h, 1.8h or 2 h) to obtain the decellularized matrix layer; the trypsin solution of the present invention may have a mass fraction of 0.25 to 0.5% (for example, 0.25%, 0.3%, 0.35%, 0.4%, 0.45% or 0.5%).
According to some preferred embodiments, the active substance-containing solution is obtained by mixing a collagen solution, epidermal Growth Factor (EGF) and basic fibroblast growth factor (bFGF);
in the solution containing the active substances, the mass fraction of collagen is 0.5-0.9%, and the concentration of the Epidermal Growth Factor (EGF) is 5ug/mL; the concentration of basic fibroblast growth factor (bFGF) is 5ug/mL.
According to some preferred embodiments, the collagen solution is obtained by mixing type I collagen with an acetic acid solution; the mass fraction of acetic acid in the acetic acid solution is 4 to 8% (for example, may be 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5% or 8%).
It should be noted that, the preparation method of the type I collagen in the present invention refers to patent CN114732954a, and includes: s1: removing fat and fascia on the beef achilles tendon with certain quality, cleaning and freezing; s2: cutting frozen beef achilles tendon to a thickness of about 0.8 mm; s3: soaking the slice cut by the S2 in sodium bicarbonate solution with the mass fraction of 1.0-1.5% for 12-24 h, and then cleaning the slice with purified water for degreasing for a plurality of times; s4: adding the sheet degreased by the step S3 into an acid solution with the pH value of 1-3, carrying out enzymolysis for 48-96 hours at the temperature of 0-25 ℃, and removing terminal peptide to obtain an enzymolysis solution; the acid solution is prepared from one or more of acetic acid, citric acid and phosphoric acid; in the invention, the acid solution with the pH value of 2 is preferable for enzymolysis, because the optimal condition for activating the added pepsin is normal temperature, the pH value is between 2 and 3, the enzymolysis is carried out for 48 to 96 hours, preferably 96 hours at normal temperature, the enzymolysis is thorough, and the yield is high; s5: centrifuging the enzymolysis liquid obtained in the step S4 by using a centrifuge, taking supernatant, and separating out collagen floccule by using a salt solution, wherein the salt is one or more of sodium chloride, potassium chloride, sodium carbonate and potassium carbonate, and the salt solution is supersaturated salt solution; s6: adding the white floccule obtained in the step S5 into a dialysis bag for dialysis, so as to ensure that the environmental change of the dialysis external liquid is mild and avoid the excessive change of the dialysis external liquid, thereby causing irreversible precipitation of collagen in the dialysis internal liquid, and finally, using purified water for dialysis to finish the purification of the collagen by adopting a gradient dialysis mode, such as gradually reducing the concentration of acetic acid in the external liquid; the manner of gradient dialysis may be, for example: firstly, placing a dialysis bag in 45L of pH=3 dialysate for dialysis for 4 days, wherein the dialysis temperature is 15+/-3 ℃, and 1 dialysate is changed every 2 days; then placing the dialysis bag in 45L of pH=4 dialysate for dialysis for 5 days, wherein the dialysis temperature is 15+/-3 ℃, and the dialysate is changed for 1 time every 1 day; placing the dialysis bag in 45L of purified water for dialysis for 6 days, wherein the dialysis temperature is 15+/-3 ℃, and the dialysate is changed for 3 times a day, once in the morning, in the middle and at night; in the present invention, the dialysate of ph=3 and the dialysate of ph=4 are, for example, acetic acid solutions prepared from acetic acid and purified water, and the preparation of the dialysate of ph=3 may be, for example: adding 45000mL of purified water into a dialysis tank, accurately taking 146.694mL of acetic acid by using a measuring cylinder and a pipetting gun, and uniformly stirring to obtain an acetic acid solution with pH=3; the formulation of the dialysate at ph=4 can be, for example: adding 45000mL of purified water into a dialysis tank, accurately taking 1.479mL of acetic acid by a liquid-transferring gun, adding into the dialysis tank, and uniformly stirring to obtain an acetic acid solution with pH=4; s7: placing the collagen gel with certain mass and solid content produced in the step S6 into a homogenizer, adding water with certain mass to prepare a collagen solution with the concentration of 0.5-0.9%, wherein the homogenizing frequency is 20-50 Hz, and the time is 15-60min; s8: and (3) freeze-drying the homogenized collagen gel in the step (S7) to obtain the type I collagen with uniform texture.
In order to more clearly illustrate the technical scheme and advantages of the present invention, the present invention will be further described below with reference to examples.
The materials and the reagents in the invention can be obtained by direct purchase or self-synthesis in the market, and the specific model is not limited.
The performance test of the examples and comparative examples of the present invention employed the following method:
antibacterial and microbial Barrier test
1) Equipment and method for manufacturing the same
(1) Test microorganism golden yellow grape ball (ATCC 6538)
(2) Culture medium, blood agar, nutrient agar and glucose nutrient broth
(3) Artificial skin: area 50 mm. Times.50 mm
2) Operating procedure
(1) The staphylococcus aureus is inoculated into 6mL glucose nutrient broth culture medium, and the bacterial suspension after 16h culture at 37 ℃ is taken as viable bacteria count.
(2) The epidermis of the artificial skin was laid flat in a sterile dish face up.
(3) By using 10 of 7 The cfu/mL staphylococcus aureus suspension is dripped on the artificial skin, and 10 drops are not contacted with each other, and each drop is 0.01mL.
(4) Placing the bacteria-infected artificial skin at the temperature of 20-25 ℃ and the relative humidity of 40-50% to dry the skin for not more than 6 hours.
(5) Spreading the bacteria-contaminated artificial skin on the surface of the blood agar culture medium, fully contacting, and removing the artificial skin after 5-6 s when the bacteria-contaminated surface is upward.
(6) Blood agar culture was performed at 37℃for 16 to 24 hours to perform colony counting.
3) Reporting the results: the number of colonies grown on each blood agar medium plate and the total number of colonies grown on 3 plates.
The release performance test method comprises the following steps: test samples (artificial skin of examples or comparative examples) with a size of 20 x 20 (mm) were placed in 10mL of simulated body fluid containing type I collagenase 0.04Unit/mL, and the simulated body fluid was periodically sampled at 37 ℃ to determine the amount of EGF and bFGF in the simulated body fluid by ELISA method, and the relationship between the release amounts of Epidermal Growth Factor (EGF) and basic fibroblast growth factor (bFGF) with time was determined.
The mechanical property testing method comprises the following steps: test specimens (the portions of the artificial skin of examples and comparative examples except for the non-absorbable layer) were prepared as rectangular test bars of 1cm×3cm, the tensile strain rate was 10mm/min, and the maximum tensile strength thereof was measured on a mechanical tester until the product was broken.
In vitro cell composite culture
1. In vitro cell implantation
(1) Cutting the artificial skin into a disc shape with the diameter of 6mm by using a puncher;
(2) Putting the prepared disc-shaped material into another culture dish, adding a proper amount of DMEM culture solution of 10% fetal calf serum to completely submerge the disc-shaped material, and incubating the disc-shaped material in a constant temperature incubator for 24 hours at 37 ℃;
(3) Mice were counted on cell counting plates after digestion of the fine fiber NIH/3T3 with 0.25% pancreatin. Centrifuging at 500rpm for 5min after cell digestion, removing supernatant, adding 5mL10% foetal calf serum DMEM culture solution, centrifuging, re-suspending, diluting cells, and adjusting concentration to 8X10 4 /mL。
(4) Taking out the materials prepared before from the incubator, placing the materials in an ultra-clean bench, clamping and transferring the materials to a 96-well plate by using sterile forceps, and placing the materials at the bottom;
(5) 100 μl of cell suspension was added to each well, each group of 4 multiplex wells (one of which was used for microscopic observation and the other three were used for detection of cell activity) were inoculated into 96-well plates according to the experimental material group, control wells containing no material in cell culture medium alone, blank wells containing no cells in culture medium alone, and cell culture in a cell incubator overnight.
(6) At 37 ℃,5% CO 2 Incubating in a constant temperature incubator, and changing liquid every 2-3 days.
CCK-8 absorbance detection
(1) To eliminate the effect of cells that do not adhere to the scaffold, the samples were transferred to a new 96-well plate.
(2) Selecting a 20mL centrifuge tube, adding 4500uL of DMEM culture solution and 500uL of CCK-8 solution, stirring, mixing, and collecting 100uL of mixed solution, wherein 90uL of DMEM culture medium and 10uL of CCK-8 solution are added into each hole, mixing, and adding 5% CO 2 Incubate for 2h at 37 ℃.
(3) The treated medium was aspirated and transferred to another 96-well plate, 100uL per well, and absorbance at 450nm was measured by an ELISA reader.
3. Microscopic examination of fibroblast and artificial skin cultures
(1) At 5 days of incubation, the medium was aspirated and washed three times with PBS (10 mm, ph=7.4).
(2) The epidermis layer and the dermis upper layer were peeled off from the artificial skin, and the cell adhesion condition on both sides of the dermis upper layer and the lower side of the epidermis layer (absorbable epidermis layer) was observed.
Example 1
An antibacterial artificial skin comprises a hypodermis layer 1, a hypodermis layer 2, an absorbable epidermis layer 3 and a non-absorbable epidermis layer 4 which are sequentially arranged from bottom to top; the thickness of the spongy porous collagen layer of the hypodermis layer 1 is 2.5mm, the aperture is 100 mu m, and the porosity is 93%; the dermis upper layer 2 is a three-layer porous acellular matrix film adsorbed with active substances, the thickness of a single-layer porous acellular matrix film is 0.1mm, the porous acellular matrix film is provided with a plurality of through holes along the thickness direction, the pore diameter of the through holes is 0.1mm, and the density is 500 pieces/cm 2 The active substance comprises Epidermal Growth Factor (EGF) and basic fibroblast growth factor (bFGF); the absorbable epidermal layer 3 is a layer of acellular matrix membrane coated with nano mesoporous bioactive glass, the thickness of a single layer acellular matrix membrane is 0.05mm, the mass ratio of the nano mesoporous bioactive glass to the acellular matrix membrane is 1:9, the particle size of the nano mesoporous bioactive glass is 40 mu m, and the aperture is 10nm; the non-absorbable skin layer 4 is a silicone rubber film with a thickness of 0.15 mm.
Example 2
An antibacterial artificial skin comprises a hypodermis layer 1, a hypodermis layer 2, an absorbable epidermis layer 3 and a non-absorbable epidermis layer 4 which are sequentially arranged from bottom to top; the thickness of the spongy porous collagen layer of the hypodermis layer 1 is 2.5mm, the aperture is 100 mu m, and the porosity is 93%; the dermis upper layer 2 is a three-layer porous acellular matrix film adsorbed with active substances, the thickness of a single-layer porous acellular matrix film is 0.1mm, the porous acellular matrix film is provided with a plurality of through holes along the thickness direction, the pore diameter of the through holes is 0.1mm, and the density is 500 pieces/cm 2 The active substance comprises Epidermal Growth Factor (EGF) andbasic fibroblast growth factor (bFGF); the absorbable epidermal layer 3 is a layer of acellular matrix membrane coated with nano mesoporous bioactive glass, the thickness of a single layer acellular matrix membrane is 0.05mm, the mass ratio of the nano mesoporous bioactive glass to the acellular matrix membrane is 1:20, the particle size of the nano mesoporous bioactive glass is 40 mu m, and the aperture is 10nm; the non-absorbable skin layer 4 is a silicone rubber film with a thickness of 0.15 mm.
Example 3
An antibacterial artificial skin comprises a hypodermis layer 1, a hypodermis layer 2, an absorbable epidermis layer 3 and a non-absorbable epidermis layer 4 which are sequentially arranged from bottom to top; the thickness of the spongy porous collagen layer of the hypodermis layer 1 is 2.5mm, the aperture is 100 mu m, and the porosity is 93%; the dermis upper layer 2 is a three-layer porous acellular matrix film adsorbed with active substances, the thickness of a single-layer porous acellular matrix film is 0.1mm, the porous acellular matrix film is provided with a plurality of through holes along the thickness direction, the pore diameter of the through holes is 0.1mm, and the density is 500 pieces/cm 2 The active substance comprises Epidermal Growth Factor (EGF) and basic fibroblast growth factor (bFGF); the absorbable epidermal layer 3 is a layer of acellular matrix membrane coated with nano mesoporous bioactive glass, the thickness of a single layer acellular matrix membrane is 0.05mm, the mass ratio of the nano mesoporous bioactive glass to the acellular matrix membrane is 1:49, the particle size of the nano mesoporous bioactive glass is 40 mu m, and the aperture is 10nm; the non-absorbable skin layer 4 is a polyvinyl alcohol film having a thickness of 0.15 mm.
Example 4
An antibacterial artificial skin comprises a hypodermis layer 1, a hypodermis layer 2, an absorbable epidermis layer 3 and a non-absorbable epidermis layer 4 which are sequentially arranged from bottom to top; the thickness of the spongy porous collagen layer of the hypodermis layer 1 is 0.5mm, the pore diameter is 20 mu m, and the porosity is 91%; the dermis upper layer 2 is a porous acellular matrix membrane adsorbed with active substances, the thickness of a single porous acellular matrix membrane is 0.02mm, the porous acellular matrix layer is provided with a plurality of through holes along the thickness direction, the pore diameter of the through holes is 1mm, and the density is 9 pieces/cm 2 The method comprises the steps of carrying out a first treatment on the surface of the The active substance comprises Epidermal Growth Factor (EGF) and basic fibroblast growth factor (bFGF); the absorbable surface layer 3 is a layerThe surface of the cell-free matrix membrane is coated with nano mesoporous bioactive glass, the thickness of a monolayer cell-free matrix membrane is 0.02mm, the mass ratio of the nano mesoporous bioactive glass to the cell-free matrix membrane is 1:9, the particle size of the nano mesoporous bioactive glass is 40 mu m, and the pore diameter is 5nm; the non-absorbable skin layer 4 is a polyvinyl alcohol film having a thickness of 0.1mm.
Example 5
An antibacterial artificial skin comprises a hypodermis layer 1, a hypodermis layer 2, an absorbable epidermis layer 3 and a non-absorbable epidermis layer 4 which are sequentially arranged from bottom to top; the thickness of the spongy porous collagen layer of the hypodermis layer 1 is 5mm, the pore diameter is 200 mu m, and the porosity is 93%; the dermis upper layer 2 is two layers of porous acellular matrix membranes adsorbed with active substances, the thickness of a single layer of porous acellular matrix membrane is 0.1mm, the porous acellular matrix layer is provided with a plurality of through holes along the thickness direction, the pore diameter of the through holes is 0.5mm, and the density is 200 pieces/cm 2 The method comprises the steps of carrying out a first treatment on the surface of the The active substance comprises Epidermal Growth Factor (EGF) and basic fibroblast growth factor (bFGF); the absorbable epidermal layer is a layer of acellular matrix membrane coated with nano mesoporous bioactive glass, the thickness of a single layer acellular matrix membrane is 0.1mm, the mass ratio of the nano mesoporous bioactive glass to the acellular matrix membrane is 1:9, the particle size of the nano mesoporous bioactive glass is 40 mu m, and the aperture is 20nm; the non-absorbable skin layer 4 was a silicone rubber film having a thickness of 0.25mm.
Comparative example 1
Comparative example 1 is substantially the same as example 1 except that: the absorbable epidermis is a layer of decellularized matrix membrane with a single layer of decellularized matrix membrane thickness of 0.1mm.
The surface of the absorbable epidermis layer is not coated with nano mesoporous bioactive glass, and the antibacterial and cell adhesion promoting abilities are weakened.
Comparative example 2
Comparative example 2 is substantially the same as example 1 except that: the mass ratio of the nano mesoporous bioactive glass to the acellular matrix membrane in the absorbable epidermal layer is 1:70.
The amount of the nano mesoporous bioactive glass coated on the surface of the absorbable epidermis layer is small, and the antibacterial and cell adhesion promoting capabilities are weakened.
Comparative example 3
Comparative example 3 is substantially the same as example 1 except that: the mass ratio of the nano mesoporous bioactive glass to the acellular matrix membrane in the absorbable epidermis layer is 1:5.
The nano mesoporous bioactive glass is excessive in dosage and is not easy to be completely spread on the surface of the acellular matrix membrane.
Comparative example 4
Comparative example 4 is substantially the same as example 1 except that: the dermis upper layer is a three-layer porous acellular matrix membrane, the thickness of the single-layer porous acellular matrix membrane is 0.1mm, the porous acellular matrix membrane is provided with a plurality of through holes along the thickness direction, the pore diameter of the through holes is 0.1mm, and the density of the through holes is 500 pieces/cm 2 。
The surface of the porous decellularized matrix membrane is not adsorbed with active substances, so that the cell proliferation capacity is weakened, and the tissue repair and reconstruction are not facilitated.
Comparative example 5
Comparative example 5 is substantially the same as example 1 except that: the dermal upper layer is three layers of acellular matrix membranes adsorbed with active substances, and the thickness of the single layer acellular matrix membrane is 0.1mm.
Since the decellularized matrix membrane is not perforated, cells cannot pass through the upper dermis and the tissue repair and reconstruction time is prolonged.
Comparative example 6
Comparative example 6 is substantially the same as example 1 except that: the porosity of the spongy porous collagen layer which is the hypodermis layer is 75%.
The porosity of the hypodermis is too small, the cell proliferation capability is weakened, and the repair and reconstruction of the hypodermis are affected.
Comparative example 7
Comparative example 7 is substantially the same as example 1 except that: no non-absorbable skin layer.
No non-absorbable epidermis layer has reduced microbial barrier capacity, increasing the risk of infection.
Table 1. Antibacterial and microbial barrier test data for artificial skin prepared in examples and comparative examples of the present invention.
As can be seen from Table 1, the absorbable surface layers of examples 1-5 and comparative examples 3-6 of the present invention are coated with the nano mesoporous bioactive glass within the dosage range of the present invention and are provided with non-absorbable surface layers, and the artificial skin surface layers have excellent antibacterial and microbial barrier properties; the comparative example 1 has poor antibacterial effect because the surface of the absorbable epidermis layer is not coated with nano mesoporous bioactive glass; the absorbable epidermis layer of comparative example 2 is coated with a small amount of nano mesoporous bioactive glass and is provided with a non-absorbable layer, so that the absorbable epidermis layer has a certain antibacterial effect; comparative example 7 was inferior in bacteriostatic effect because of no non-absorbable skin layer; therefore, the nano mesoporous bioactive glass is coated on the surface of the absorbable epidermis layer, and meanwhile, the non-absorbable epidermis layer is arranged to ensure that the artificial skin has excellent antibacterial and microorganism barrier properties.
Table 2. Release process data of Epidermal Growth Factor (EGF) and basic fibroblast growth factor (bFGF) of artificial skin (excluding non-absorbable epidermis layer) prepared in examples and comparative examples of the present invention.
As can be seen from Table 2, the release of Epidermal Growth Factor (EGF) and basic fibroblast growth factor (bFGF) in the upper dermis of the antibacterial artificial skin is faster in the first days, and the release is slower in the later days, so that the antibacterial artificial skin can be well matched with natural wound repair and can promote the skin repair process.
Table 3. Mechanical test data for artificial skin (excluding non-absorbable skin layers) prepared in examples and comparative examples of the present invention.
As can be seen from Table 3, the bacteriostatic artificial skin of the present invention has good mechanical properties.
Table 4. The experimental data of the in vitro cell composite culture of the artificial skin prepared in the examples and comparative examples of the present invention.
It should be noted that the number of the substrates, in the table "+++" indicates a large number of cells are grown and, "++" indicates small cell growth, "+" indicates very small cell growth, "-" no cell growth: the inner surface of the absorbable epidermis layer is one surface close to the dermis upper layer, and the outer surface of the dermis upper layer is one surface close to the absorbable epidermis layer; the inner surface of the dermis upper layer is one surface close to the dermis lower layer; the magnitude of the OD reflects the strength of the proliferation-promoting ability of the cells, and the larger the OD, the stronger the proliferation-promoting ability of the cells.
Examples 1-5 compared with comparative examples, it is clear that in comparative examples 1-2, the surface of the absorbable epidermis layer is not or little coated with the nano mesoporous bioactive glass, and the lower surface of the absorbable epidermis layer has only a very small amount of cells growing; comparative example 4 the surface of the porous decellularized matrix membrane in the upper dermis layer did not adsorb active substances, and the cell proliferation capacity was reduced, and only a very small amount of cells grew on both the lower surface of the epidermis layer and the upper surface of the upper dermis layer were absorbable; comparative example 5 the decellularized matrix membrane in the upper dermis was not perforated and cells were unable to pass through the upper dermis and the lower surface of the absorbable epidermis grew without cells; the porosity of the lower dermis layer in comparative example 6 was too small, the cell proliferation ability was reduced, the upper surface of the upper dermis layer had only a very small amount of cells grown, and the lower surface of the absorbable epidermis layer had only a very small amount of cells grown.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. An antibacterial artificial skin is characterized by comprising a hypodermis layer, a hypodermis upper layer, an absorbable epidermis layer and a non-absorbable epidermis layer which are sequentially arranged from bottom to top;
the hypodermis layer is a spongy porous collagen layer;
the dermis upper layer is a porous acellular matrix layer; the porous decellularized matrix layer at least comprises a porous decellularized matrix film adsorbed with active substances; the active substance comprises an epidermal growth factor and an alkaline fibroblast growth factor;
the absorbable epidermis layer is a bacteriostatic decellularized matrix layer; the antibacterial acellular matrix layer comprises an acellular matrix film with a surface coated with nano mesoporous bioactive glass;
the non-absorbable skin layer is a film layer.
2. The antimicrobial artificial skin according to claim 1, wherein the spongy porous collagen layer is obtained by freeze-drying a collagen solution;
the thickness of the spongy porous collagen layer is 0.5-5 mm;
the pore diameter of the spongy porous collagen layer is 20-200 mu m; the spongy porous collagen layer has a porosity of greater than 90%.
3. The antimicrobial artificial skin according to claim 1, wherein the thickness of the porous decellularized matrix film is 0.02 to 0.1mm;
the porous acellular matrix membrane is provided with a plurality of through holes along the thickness directionThe diameter of the through holes is 0.1-1 mm, and the density of the through holes is 9-900/cm 2 。
4. The antimicrobial artificial skin according to claim 1, wherein the thickness of the decellularized matrix film is 0.02 to 0.1mm;
the mass ratio of the nano mesoporous bioactive glass to the acellular matrix membrane is 1 (9-49).
5. The antimicrobial artificial skin according to claim 1, wherein the nano mesoporous bioactive glass has a particle size of not more than 50 μm and a pore size of 5 to 20nm.
6. The antimicrobial artificial skin according to claim 1, wherein the non-absorbable skin layer is a silicone rubber film or a polyvinyl alcohol film; the thickness of the non-absorbable skin layer is 0.1-0.25 mm.
7. A method of preparing an antimicrobial artificial skin according to any one of claims 1-6, comprising the steps of:
s1, cleaning fresh tissues, and then carrying out disinfection, degreasing and decellularization treatment to obtain a decellularized matrix membrane;
s2, freeze-drying and perforating the acellular matrix membrane, and then soaking the acellular matrix membrane in a solution containing active substances to obtain a porous acellular matrix membrane adsorbed with the active substances, namely an upper acellular matrix membrane of dermis;
s3, the nanometer mesoporous bioactive glass is spread on the surface of the acellular matrix film, so that the acellular matrix film with the surface coated with the nanometer mesoporous bioactive glass is obtained, and the acellular matrix film of the epidermis layer can be absorbed;
s4, sequentially paving the absorbable epidermis layer acellular matrix membrane, the dermis upper layer acellular matrix membrane and the collagen solution in a mould, and compositing a non-absorbable epidermis layer on the absorbable epidermis layer after freeze drying to obtain the antibacterial artificial skin.
8. The preparation method according to claim 7, wherein in step S1, the sterilization is to soak the fresh tissue after washing in an ethanol solution of peracetic acid for 30 to 60 minutes;
the degreasing is to soak the sterilized tissue in sodium hydroxide solution for 30-60 min, and then clean the tissue with phosphate buffer;
the decellularized treatment is to soak the defatted tissue in trypsin solution for 1-2 h to obtain the decellularized matrix layer.
9. The method according to claim 7, wherein the active material-containing solution is obtained by mixing a collagen solution, an epidermal growth factor and an alkaline fibroblast growth factor;
in the solution containing the active substances, the mass fraction of collagen is 0.5-0.9%, and the concentration of the epidermal cell growth factor is 5ug/mL; the concentration of the basic fibroblast growth factor is 5ug/mL.
10. A method of preparation according to claim 9, wherein the collagen solution is obtained by mixing type I collagen with an acetic acid solution; the mass fraction of acetic acid in the acetic acid solution is 4-8%.
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