CN114732954B - Medicine-carrying type artificial skin and preparation method thereof - Google Patents

Medicine-carrying type artificial skin and preparation method thereof Download PDF

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Publication number
CN114732954B
CN114732954B CN202210369287.XA CN202210369287A CN114732954B CN 114732954 B CN114732954 B CN 114732954B CN 202210369287 A CN202210369287 A CN 202210369287A CN 114732954 B CN114732954 B CN 114732954B
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layer
collagen
dermis
silk fibroin
solution
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CN114732954A (en
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胡艳丽
宋天喜
崔云
朱金亮
何志敏
胡刚
仇志烨
吴晶晶
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Shandong Aojing Biotechnology Co ltd
Aojing Medical Technology Co ltd
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Shandong Aojing Biotechnology Co ltd
Aojing Medical Technology Co ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/60—Materials for use in artificial skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body
    • A61F2/10—Hair or skin implants
    • A61F2/105—Skin implants, e.g. artificial skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/24—Collagen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/56—Porous materials, e.g. foams or sponges
    • D—TEXTILES; PAPER
    • D04—BRAIDING; LACE-MAKING; KNITTING; TRIMMINGS; NON-WOVEN FABRICS
    • D04H—MAKING TEXTILE FABRICS, e.g. FROM FIBRES OR FILAMENTARY MATERIAL; FABRICS MADE BY SUCH PROCESSES OR APPARATUS, e.g. FELTS, NON-WOVEN FABRICS; COTTON-WOOL; WADDING ; NON-WOVEN FABRICS FROM STAPLE FIBRES, FILAMENTS OR YARNS, BONDED WITH AT LEAST ONE WEB-LIKE MATERIAL DURING THEIR CONSOLIDATION
    • D04H1/00—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres
    • D04H1/04—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres from fleeces or layers composed of fibres having existing or potential cohesive properties, e.g. natural fibres, prestretched or fibrillated artificial fibres
    • D04H1/30—Collagen
    • D—TEXTILES; PAPER
    • D04—BRAIDING; LACE-MAKING; KNITTING; TRIMMINGS; NON-WOVEN FABRICS
    • D04H—MAKING TEXTILE FABRICS, e.g. FROM FIBRES OR FILAMENTARY MATERIAL; FABRICS MADE BY SUCH PROCESSES OR APPARATUS, e.g. FELTS, NON-WOVEN FABRICS; COTTON-WOOL; WADDING ; NON-WOVEN FABRICS FROM STAPLE FIBRES, FILAMENTS OR YARNS, BONDED WITH AT LEAST ONE WEB-LIKE MATERIAL DURING THEIR CONSOLIDATION
    • D04H1/00—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres
    • D04H1/70—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres characterised by the method of forming fleeces or layers, e.g. reorientation of fibres
    • D04H1/72—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres characterised by the method of forming fleeces or layers, e.g. reorientation of fibres the fibres being randomly arranged
    • D04H1/728—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres characterised by the method of forming fleeces or layers, e.g. reorientation of fibres the fibres being randomly arranged by electro-spinning
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/404—Biocides, antimicrobial agents, antiseptic agents
    • A61L2300/406—Antibiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/60—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special physical form
    • A61L2300/602—Type of release, e.g. controlled, sustained, slow

Abstract

The invention relates to a drug-loaded artificial skin and a preparation method thereof. The drug-loaded artificial skin comprises an epidermal layer and a dermal layer; the epidermal layer is formed by compounding collagen, silk fibroin and mupirocin; the dermis comprises an upper dermis layer and a lower dermis layer, the upper dermis layer is a fine fiber structure layer composed of collagen and silk fibroin, and the lower dermis layer is a coarse fiber structure layer composed of collagen and silk fibroin. The drug-loaded artificial skin provided by the invention comprises a surface layer mixed with mupirocin antibacterial drugs, and the surface layer has a microporous structure, so that the membrane has a ventilation function while resisting bacteria, resisting bacteria and preventing water and liquid from overflowing from a wound surface; the dermis layer solves the problem of disorder porous structure existing in the existing artificial skin by adjusting the diameter and the pore diameter of the spinning, is convenient for fibroblasts and capillaries to grow into a three-dimensional structure, and under the condition of sufficient blood supply, the grown cells keep high activity, secrete collagen protein, and the dermis can be reconstructed.

Description

Medicine-carrying type artificial skin and preparation method thereof
Technical Field
The invention belongs to the technical field of biomedical materials, and particularly relates to a drug-loaded artificial skin and a preparation method thereof.
Background
The skin is the largest organ of the human body, the skin accounts for about 16 percent of the weight of the human body, the structure of the skin is composed of epidermis, dermis and subcutaneous tissues, the skin has the functions of protection, secretion, perception and metabolism, skin deficiency is a common tissue injury disease, but the skin of the human body is a tissue with strong regeneration capacity, even if large-area injury exists, the skin can be gradually recovered due to treatment, but large-area burn or wound cannot be repaired by the skin, and the artificial skin is required to be used as a temporary wound surface protection covering material in the treatment process to help.
Autologous skin grafting, allogeneic skin grafting and xenogeneic skin grafting are common skin defect repair approaches, but have the defects of insufficient supply area, immunological rejection, disease transmission and the like; chinese invention patent CN101143231B discloses a tissue engineering skin containing myocytes and a preparation method thereof, which comprises the steps of digesting the taken tissue, separating into epidermal cells and fiber cells, performing separation culture and amplification on the epidermal cells and the fiber cells, recombining into a double-layer artificial skin tissue after reaching a certain quantity, wherein the artificial skin raw material is taken from human tissues, has certain risk and is expensive and is hardly accepted by general patients. Therefore, how to develop artificial skin with structure and function similar to normal skin of human body as substitute of injured skin, playing the role of protecting wound and promoting wound healing is a problem to be solved in clinic, and the artificial skin is easy to preserve and transport, has reliable technology, can be produced in batch, is safe in production and mature in quality control, and occupies the mainstream of market development.
The artificial skin can be divided into artificial epidermis, artificial dermis and composite skin with epidermis and dermis, the artificial epidermis transplantation can prevent the liquid loss of the wound surface and block the invasion of external microorganisms, but normal anchoring fibers cannot be formed on the deep wound surface due to the lack of a dermis structure, so that the transplantation survival rate is low, the wound surface contracture is obvious, and the anti-infection capability is poor. The artificial dermis is implanted into the full-layer skin defect wound surface, can be quickly adhered to the wound surface, has abundant blood vessels growing in, but has poor anti-infection capability and liquid preservation capability due to the lack of epidermal components, and usually needs to be implanted with autologous skin for the second time after the artificial dermis is implanted. The composite skin has epidermal layer and dermal layer, and after the composite skin is implanted into the damaged part, the semi-permeable epidermal layer can protect, ventilate and prevent bacterial invasion, and the dermal layer has three-dimensional porous rack structure. The dermis layer of the commercial composite artificial skin consists of sponge prepared by covalently crosslinking collagen and 6-chondroitin sulfate, and the epidermis layer is a silica gel membrane, so that although the mechanical property of the composite porous scaffold of the dermis layer is slightly improved, the artificial skin has the defects that the degradation period is difficult to control, the porous structure is irregular and the like due to larger differences of the structures, the properties and the like of the collagen and the 6-chondroitin sulfate; for example, the chinese patent application CN107050521a discloses a double-layer collagen dermal scaffold and a preparation method thereof, wherein the epidermal layer is a silica gel membrane, the dermal layer is a collagen-chondroitin sulfate composite scaffold, and the defect of disordered porous structure of the dermal layer also exists.
In addition, in the clinical treatment process, bacterial infection is a big problem for patients, researches show that glucose bacteria and streptococcus are main bacteria causing skin infection, a common method for treating bacterial infection is to inject antibiotics in a systemic administration mode to cause bacterial drug resistance, and local administration is a good choice, so that the problems of disordered porous structures, vascularization, wound infection and the like of the existing artificial skin are faced, and the artificial skin with better physical properties and antibacterial properties is urgently needed to be provided for treating the skin wound.
Collagen is the main ingredient in human skin, collagen accounts for 72% in the skin, collagen 80% in the dermis, a large number of biological and physical research results show that collagen has active biological function, can actively participate in migration, differentiation and proliferation of cells, can be in direct contact with blood, can improve the microenvironment of epidermal cells, promote the synthesis of amino acid necessary for metabolism of skin tissues, and accelerate skin regeneration and repair. Collagen has good biocompatibility, biodegradability, low immunogenicity, low cytotoxicity, cell growth promotion and other properties, and is widely used for artificial skin development, but the collagen has the defects of inelasticity, low strength and high degradation speed in vivo, so that the application of the collagen is limited.
The silk fibroin is a natural fiber protein, contains 18 amino acids, 11 of which are essential amino acids for human bodies, has no toxic action on human bodies, is safe and reliable, has good biological activity, mechanical property and physicochemical property, belongs to fiber proteins with similar structures as the skin proteins of the human bodies, and the affinity of the silk fibroin and hydrolysate thereof to the skin of the human bodies is incomparable with that of any other natural protein. Compared with collagen, the silk fibroin has good biocompatibility and sufficient mechanical strength, the slow degradation speed of the silk fibroin can provide long-term support for cell growth, and the composite material of the silk fibroin and the collagen integrates the advantages of two materials, overcomes the defect of the application of a single material, increases the mechanical strength of a collagen-based product, relieves the degradation rate of the collagen-based product, and can regulate the degradation rate. However, no report is found on artificial skin which simultaneously adopts collagen and silk fibroin to form an epidermal layer and a dermal layer and has excellent performance.
In summary, there is a need for a novel drug-loaded artificial skin and a method for preparing the same.
Disclosure of Invention
The invention provides a drug-loaded artificial skin and a preparation method thereof, aiming at solving one or more technical problems in the prior art. The invention provides a drug-loaded artificial skin, which is a drug-loaded artificial tissue engineering skin consisting of type I collagen, silk fibroin and mupirocin, and is provided with an epidermal layer and a dermal layer, wherein the epidermal layer is bionic to the greatest extent in composition and structure, consists of collagen and silk fibroin and is mixed with mupirocin antibacterial drugs, and the epidermal layer has a microporous structure, so that the membrane has a ventilation function while resisting bacteria and preventing water and liquid from overflowing from a wound surface; the dermis is a stepped mesh structure obtained by adopting a spinning technology through collagen and silk fibroin, the diameter and the aperture of the spinning are adjusted through the electrospinning technology, the problem that the porous structure of the existing artificial skin is disordered is solved, the fibroblasts and capillaries can conveniently grow into a three-dimensional structure, the growing cells keep high activity under the condition of sufficient blood supply, the cells secrete the collagen, and the dermis can be reconstructed.
The invention provides a drug-loaded artificial skin in a first aspect, which comprises an epidermal layer and a dermal layer; the epidermal layer is formed by compounding collagen, silk fibroin and mupirocin; the dermis comprises an upper dermis layer and a lower dermis layer, the upper dermis layer is a fine fiber structure layer composed of collagen and silk fibroin, and the lower dermis layer is a coarse fiber structure layer composed of collagen and silk fibroin.
Preferably, the drug-loaded artificial skin has one or more of the following properties:
the thickness of the surface layer is 0.07-1.2 mm;
the thickness of the corium layer is 2-4mm, and the thickness of the upper corium layer is 1-2 mm;
the fiber diameter of the upper corium layer is 580-647nm, and the porosity is 50-60%;
the fiber diameter of the lower corium layer is 700-780nm, and the porosity is 65-80%;
the upper dermal layer is closer to the epidermal layer than the lower dermal layer;
the skin layer has a micropore structure, the micropore structure is formed by uniformly distributing a plurality of micropores, and the aperture of each micropore is preferably 60-110 μm;
the surface of the epidermal layer, which is close to the upper dermal layer, is rough, the surface of the epidermal layer, which is far away from the upper dermal layer, is smooth, and the rough surface of the epidermal layer is provided with a reticular structure;
the upper dermis layer and the lower dermis layer are both prepared by an electrospinning technology.
The invention provides a preparation method of a drug-loaded artificial skin in a second aspect, the drug-loaded artificial skin comprises an epidermal layer and a dermal layer, the dermal layer comprises an upper dermal layer and a lower dermal layer, and the method comprises the following steps:
(1) Dissolving mupirocin in water to obtain mupirocin solution;
(2) Adding silk fibroin and collagen into an acetic acid solution and uniformly stirring to obtain a silk fibroin-collagen solution;
(3) Mixing the mupirocin solution and the silk fibroin-collagen solution and uniformly stirring to obtain a mixed solution;
(4) Injecting the mixed solution into a mold, carrying out pore-forming by a template method, and then drying, demolding and crosslinking to obtain a skin layer;
(5) Adding collagen and silk fibroin into hexafluoroisopropanol or acetic acid solution to prepare a first spinning solution with the concentration of 4-6% and a second spinning solution with the concentration of 7-9%;
(6) Preparing a lower dermis layer by adopting the second spinning solution through an electrospinning technology, preparing an upper dermis layer on the basis of the lower dermis layer by adopting the first spinning solution through the electrospinning technology, and then preparing a dermis layer through crosslinking;
(7) Coating acetic acid solution of collagen on the epidermal layer, then covering the epidermal layer on the upper dermal layer of the dermal layer, and then carrying out freeze drying and crosslinking to prepare the drug-loaded artificial skin.
Preferably, one side of the skin layer prepared in the step (4) is smooth, and the other side is rough; in the step (7), a collagen acetic acid solution is coated on the rough surface of the epidermis layer, and then the epidermis layer including the upper dermis layer is covered, and then the drug-loaded artificial skin is prepared by freeze drying and crosslinking.
Preferably, in the step (1), the mupirocin solution contains mupirocin with a mass fraction of 0.5-1.5%, preferably 1%; in the step (2), the mass ratio of the silk fibroin to the collagen is 1: (0.5-2), wherein the concentration of the silk fibroin-collagen solution is 4-7%; in the step (4), the bottom end of the mold is provided with micropores which are formed by laser, copper wires with the diameter of 50-100 μm are inserted into the micropores, a non-woven fabric net is inserted into the bottom of the copper wires, and the non-woven fabric net is flatly laid at the bottom end of the mold, preferably, the non-woven fabric net is a non-woven fabric net with 140-300 meshes; in the step (5), the mass ratio of the collagen to the silk fibroin is 1: (0.5-2), preferably 2:1, the concentration of the first spinning solution is 6% and the concentration of the second spinning solution is 8%; in step (4), step (6) and/or step (7), the crosslinking is: ultraviolet irradiation is carried out for 12-48 h; and/or in the step (7), the collagen acetic acid solution contains 4 to 8 percent of collagen by mass, and preferably 5 percent of collagen by mass.
Preferably, the electrospinning parameters for preparing the upper and/or lower dermis layers are: the voltage is 15-25 kV, the receiving distance is 10-17 cm, and the flow rate of the first spinning solution and/or the second spinning solution is 1-3 mL/h.
Preferably, the mass fraction of acetic acid contained in the acetic acid solution is 4-8%, preferably 5%; and/or the collagen is type I collagen.
Preferably, in the step (2), the stirring time is 2-4 h; and/or in the step (3), the stirring time is 1-3 h.
In a third aspect, the invention provides the drug-loaded artificial skin prepared by the preparation method in the second aspect, wherein the epidermal layer is formed by compounding collagen, silk fibroin and mupirocin; the upper dermis is a fine fiber structure layer composed of collagen and silk fibroin, and the lower dermis is a coarse fiber structure layer composed of collagen and silk fibroin.
Preferably, the drug-loaded skin has one or more of the following properties:
the thickness of the surface layer is 0.07-1.2 mm;
the skin layer has a micropore structure, the micropore structure is formed by uniformly distributing a plurality of micropores, and the aperture of each micropore is preferably 60-110 μm;
one rough surface of the epidermal layer is provided with a net structure;
the thickness of the corium layer is 2-4mm, and the thickness of the upper corium layer is 1-2 mm;
the fiber diameter of the upper corium layer is 580-647nm, and the porosity is 50-60%;
the fiber diameter of the lower corium layer is 700-780nm, and the porosity is 65-80%.
Compared with the prior art, the invention at least has the following beneficial effects:
(1) The invention provides a drug-loaded artificial skin, which is provided with an epidermal layer and a dermal layer, wherein in some preferred embodiments, the thickness of the epidermal layer is 0.07-1.2mm, which is similar to the thickness of the epidermal layer of human skin, one surface of the membrane is smooth and the other surface is rough, the rough surface is similar to the mastoid layer on the surface of the dermal layer of human skin, so that the composite with the dermal layer is facilitated, the epidermal layer is a semi-transparent film with a porous structure formed by a self-made mould by adopting a template method, and the membrane has the function of ventilation while resisting bacteria, preventing water and liquid from overflowing from the wound surface; compared with the epidermal layer of the silicon membrane on the market, the epidermal layer in the invention does not need to be taken down for the second time, so that the harm to patients caused by the secondary taking down is avoided; the epidermis layer carries mupirocin antibacterial drugs, the mupirocin is mainly used for skin infection caused by gram-positive cocci, and the mupirocin is gradually released along with the degradation of the epidermis layer, so that the artificial skin is endowed with an antibacterial function, and the risk of secondary infection of a patient is avoided; the invention adds the corresponding mupirocin medicament to the epidermal layer of the artificial skin by a mixing and dissolving method, thereby effectively preventing infection complications in the treatment process and further relieving the pain of a patient in the treatment process.
(2) The dermis layer of the skin is composed of a papillary layer and a reticular layer, the papillary layer keeps a wavy shape to ensure the elasticity, capillary, lymph gland and nerve distribution of the skin, the reticular layer is mainly an elastic structure composed of coarse collagen fibers, elastic fibers and reticular fibers, the dermis layer of the drug-loaded artificial skin simulates the dermis layer of a human body and has a double-layer structure, in some preferred embodiments, the membrane thickness is 2-4mm, the upper layer is a fine fiber structure composed of collagen and silk fibroin, the fiber diameter is 580-647nm, the porosity is 50-60%, the fiber diameter of the lower layer is 700-780nm, and the porosity is 65-80%, so that the reticular structure of the dermis layer of the human body is effectively simulated, fibroblasts and capillaries can grow into a three-dimensional structure conveniently, and under the condition of sufficient blood supply, the growing cells keep high activity, and secrete collagen and reconstruct the dermis.
(3) According to the invention, the contact surface of the epidermal layer and the dermal layer of the drug-loaded artificial skin adopts the acetic acid solution of collagen as the adhesive, so that the epidermal layer and the dermal layer are effectively adhered together, and the risk of introducing biogel is avoided; the epidermis layer and the dermis layer of the drug-loaded artificial skin are physically crosslinked, so that the introduction of a crosslinking agent is avoided, and the safety of the artificial skin is improved.
(4) The artificial skin prepared by the method has no immunological rejection, low cost and wide raw material source, is used for covering a skin defect area to be used as a temporary substitute for epidermis and dermis, can keep the wound surface clean, effectively control pain and induce autologous skin regeneration and repair.
Drawings
Fig. 1 is a process flow diagram for preparing silk fibroin in some embodiments of the present invention.
FIG. 2 is a flow chart of a process for preparing collagen according to some embodiments of the present invention.
Fig. 3 is a process flow diagram for preparing the epidermal layer of the drug-loaded artificial skin in some embodiments of the present invention.
FIG. 4 is a flow chart of a process for preparing the drug-loaded artificial skin according to some embodiments of the present invention.
Fig. 5 is a schematic diagram of the internal structure of a mold used to prepare the skin layer of the present invention.
Fig. 6 is an enlarged schematic view of the bottom end of the mold of fig. 5.
In fig. 5 and 6: 1: a nonwoven web; 2: micropores; 3: and (3) copper wires.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
The invention provides a drug-loaded artificial skin in a first aspect, which comprises an epidermal layer and a dermal layer; the skin layer is formed by compounding collagen (such as type I collagen), silk fibroin and mupirocin, preferably, the skin layer has a micropore structure, the micropore structure is formed by uniformly distributing a plurality of micropores, the pore diameter of each micropore is 60-110 microns, preferably, the micropores are distributed in an array manner, the pore diameter of each micropore is independently 60-110 microns, the pore diameter of each micropore can be the same or different, and the skin layer is a drug-loaded, perforated and semitransparent artificial skin (semitransparent film) formed by compounding type I collagen, silk fibroin and mupirocin; in the invention, the epidermal layer consists of degradable collagen, silk fibroin and mupirocin, compared with the existing commercially available silica gel epidermal layer, the damage to a patient caused by secondary taking-down is avoided, mupirocin capable of treating skin infection caused by gram-positive cocci is carried, and the mupirocin is gradually released along with the degradation of the epidermal layer, so that the artificial skin disclosed by the invention has antibacterial performance; the dermis layers comprise an upper dermis layer and a lower dermis layer, the upper dermis layer is a fine fiber structure layer consisting of collagen (such as type I collagen) and silk fibroin, and the lower dermis layer is a coarse fiber structure layer consisting of collagen (such as type I collagen) and silk fibroin, so that the dermis layer is of a stepped mesh structure; in the invention, the fine fiber structure layer and the coarse fiber structure layer are relatively, preferably, the fine fiber structure layer is a fine fiber structure layer with fiber diameter of 580-647nm and porosity of 50-60%, and the coarse fiber structure layer is a coarse fiber structure layer with fiber diameter of 700-780nm and porosity of 65-80%; in the present invention, preferably, the dermis is a stepped mesh structure obtained by electrospinning collagen and silk fibroin, which facilitates the growth of fibroblasts and capillaries into a three-dimensional structure, and under the condition of sufficient blood supply, the grown cells maintain high activity, and secrete collagen, so that the dermis is reconstructed; in addition, the drug-loaded artificial skin contains silk fibroin, so that the mechanical strength of collagen can be effectively improved, and the drug-loaded artificial skin has similar softness to skin.
According to some preferred embodiments, the drug-loaded artificial skin has one or more of the following properties:
the thickness of the skin layer is 0.07 to 1.2mm (e.g., 0.07, 0.08, 0.09, 0.1, 0.12, 0.15, 0.18, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1.0, 1.1, or 1.2 mm); the thickness of the dermis layer is 2-4mm (e.g., 2, 2.5, 3, 3.5 or 4 mm), and the thickness of the upper dermis layer is 1-2mm (e.g., 1, 1.2, 1.5, 1.8 or 2 mm); the fiber diameter of the upper corium layer is 580-647nm, and the porosity is 50-60%; the fiber diameter of the lower corium layer is 700-780nm, and the porosity is 65-80%; the upper dermal layer is closer to the epidermal layer than the lower dermal layer; the skin layer has a micropore structure, the micropore structure is formed by uniformly distributing a plurality of micropores, and the aperture of each micropore is preferably 60-110 μm; in the invention, the epidermis layer has a micropore structure along the thickness direction, the side of the epidermis layer close to the upper dermis layer is rough (relatively rough), the side of the epidermis layer away from the upper dermis layer is smooth (relatively smooth), and the rough side of the epidermis layer has a reticular structure; the upper corium layer and the lower corium layer are both prepared by an electrospinning technology; the invention does not require any particular roughness or smoothness of the two sides of the skin layer, both roughness and smoothness being relative.
According to some preferred embodiments, the dermis layer has a double-layer structure, the thickness of the dermis layer is 2-4mm, the upper dermis layer is a fine fiber structure composed of collagen and silk fibroin, the fiber diameter is 580-647nm, the porosity is 50-60%, the lower dermis layer is a coarse fiber structure composed of collagen and silk fibroin, the fiber diameter is 700-780nm, and the porosity is 65-80%, so that the reticular structure of the dermis layer of a human body is effectively simulated, fibroblasts and capillaries can grow into a three-dimensional structure, grown cells can keep high activity under the condition of sufficient blood supply, the cells secrete collagen, and the dermis can be reconstructed.
The invention provides a preparation method of a drug-loaded artificial skin in a second aspect, the drug-loaded artificial skin comprises an epidermal layer and a dermal layer, the dermal layer comprises an upper dermal layer and a lower dermal layer, and the method comprises the following steps:
(1) Dissolving mupirocin in water (purified water) to obtain mupirocin solution;
(2) Adding silk fibroin and collagen into an acetic acid solution and uniformly stirring to obtain a silk fibroin-collagen solution; the invention has no particular limitation on the sources of the silk fibroin and the type I collagen;
(3) Mixing the mupirocin solution and the silk fibroin-collagen solution and uniformly stirring to obtain a mixed solution;
(4) Injecting the mixed solution into a mold, performing pore-forming by a template method, and then drying, demolding and crosslinking to obtain a skin layer; preferably, one side of the prepared skin layer is smooth, and the other side of the prepared skin layer is rough; in the present invention, both smooth and rough are relative; in the present invention, the drying process may be, for example, directly drying the mold injected with the mixed solution at normal temperature (for example, 20 to 30 ℃) (drying time is, for example, not less than 10 hours), which may ensure obtaining a translucent epidermis layer, and the translucent epidermis layer may monitor the healing of the wound;
(5) Adding collagen and silk fibroin into hexafluoroisopropanol or acetic acid solution (such as acetic acid solution with mass concentration of 4-8%), and preparing a first spinning solution with concentration of 4-6% and a second spinning solution with concentration of 7-9%;
(6) Preparing a lower dermis layer by adopting the second spinning solution through an electrospinning technology, preparing an upper dermis layer on the basis of the lower dermis layer by adopting the first spinning solution through the electrospinning technology, and then preparing a dermis layer through crosslinking;
(7) Coating acetic acid solution of collagen on the epidermal layer, then covering the epidermal layer on the upper dermal layer of the dermal layer, and then carrying out freeze drying and crosslinking to prepare the drug-loaded artificial skin; the invention has no special requirement on the thickness of the acetic acid solution for coating the collagen on the epidermal layer, and a thin layer is coated; the conditions of the freeze-drying are not particularly limited in the present invention, and the freeze-drying may be performed by using the conventional freeze-drying conditions, and preferably, the freeze-drying may be performed according to the freeze-drying conditions in table 1, wherein the freeze-drying includes a prefreezing stage, a first sublimation stage, and a second sublimation stage, and the target temperature and duration of each stage are shown in table 1 below.
Table 1: and (4) freeze-drying conditions.
Figure BDA0003587311250000101
Figure BDA0003587311250000111
In the present invention, preferably, the rough surface of the epidermis layer is coated with an acetic acid solution of collagen, and then the epidermis layer including the upper dermis layer is covered with the acetic acid solution, and then the drug-loaded artificial skin is prepared by freeze-drying and crosslinking.
According to some preferred embodiments, one side of the skin layer produced in step (4) is smooth and the other side is rough; in the step (7), a collagen acetic acid solution is coated on the rough surface of the epidermis layer, and then the epidermis layer including the upper dermis layer is covered, and then the drug-loaded artificial skin is prepared by freeze drying and crosslinking.
According to some preferred embodiments, in step (1), the mupirocin solution contains mupirocin in a mass fraction of 0.5 to 1.5% (e.g. 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4% or 1.5%), preferably 1%; in the step (2), the mass ratio of the silk fibroin to the collagen is 1: (0.5-2) (e.g., 1.5, 1, 0.8, 1:1, 1.2, 1.5, 1.8, or 1:2), the concentration of the silk fibroin-collagen solution is 4-7% (e.g., 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, or 7%); in the invention, the concentration of the silk fibroin-collagen solution is 4-7%, which means that the total mass fraction of silk fibroin and collagen contained in the silk fibroin-collagen solution is 4-7%; the invention finds that the mechanical property of the product can be improved by adding silk fibroin, but the degradation time of the product can be influenced by adding silk fibroin too much, and in the invention, the mass ratio of the silk fibroin to the collagen is preferably 1: (0.5-2), the invention discovers through in vitro degradation test that when the mass ratio of the silk fibroin and the collagen is 1:2, the mechanical property and the degradation time can better meet the requirements of artificial skin on the basic mechanical property and the degradation time.
According to some preferred embodiments, in step (4), the bottom end (bottom) of the mold (inside of the mold) has a plurality of laser-drilled micropores (the diameter of the micropores is 60-110 μm), a plurality of micropores do not penetrate through the bottom of the mold, a plurality of micropores are distributed at the bottom of the mold, for example, in an array, each micropore is inserted with a copper wire with a diameter of 50-100 μm and a length of, for example, 5cm, and the bottom of the copper wire is inserted with a nonwoven web, the nonwoven web is laid on the bottom end of the mold, and preferably, the nonwoven web is a nonwoven web with a mesh size of 140-300 meshes; in the present invention, the nonwoven fabric net is disposed such that one end (one side) of the skin layer is a rough surface having a net structure and the other end (the other side) of the skin layer is a smooth surface due to the mold not being provided with the nonwoven fabric net; in the invention, a template method is adopted, and the special mould is preferably used for pore forming, so that the epidermal layer of the drug-loaded artificial skin disclosed by the invention can endow the membrane with a ventilation function while resisting bacteria and preventing water and liquid from overflowing from a wound surface; the invention adopts a template method, carries out pore-forming by the special mould, and the prepared epidermal layer has a smooth surface and a rough surface, the rough surface is similar to the mastoid layer on the surface of the human skin dermal layer, and the physical crosslinking is carried out, thereby improving the strength, delaying the degradation speed and avoiding the toxicity generated by chemical crosslinking. In the invention, the schematic diagram of the interior of the mold for preparing the skin layer is shown in fig. 5, a layer of non-woven fabric net is laid at the bottom end of the mold, and a plurality of thin copper wires are inserted; the bottom enlarged schematic view of the die is shown in fig. 6, all inserted copper wires are not shown in fig. 6, fig. 6 is provided for illustrative purposes only, and the proportion, the size and the number of parts in the drawing are not necessarily consistent with those of an actual product; in the invention, preferably, the aperture of the micropores is 60-110 μm, the diameter of the copper wire is 50-100 μm, and the non-woven fabric net is 140-300 meshes; in the invention, it is more preferable that the mesh aperture size of the non-woven fabric net is matched with the diameter size of the copper wire, so that the copper wire can be conveniently inserted, and on the other hand, if the mesh aperture size of the non-woven fabric net is larger than the diameter size of the copper wire too much, the mold filling liquid can penetrate through the non-woven fabric during molding, and molding is carried out between the non-woven fabric and the mold copper wire.
According to some preferred embodiments, in step (5), the mass ratio of collagen to silk fibroin is 1: (0.5-2) (e.g. 1.5, 1, 0.8, 1:1, 1.2, 1.5, 1.8 or 1:2), preferably 2:1, which is more advantageous for spinning solution filamentation, whereas if silk fibroin content is relatively higher than collagen content, the spinning solution is less prone to filamentation; the concentration of the first spinning solution is 6% and the concentration of the second spinning solution is 8%, and in the present invention, the concentration of the first spinning solution refers to the total mass fraction of collagen and silk fibroin contained in the first spinning solution, and similarly, the concentration of the second spinning solution refers to the total mass fraction of collagen and silk fibroin contained in the second spinning solution; in step (4), step (6) and/or step (7), the crosslinking is: the ultraviolet irradiation is carried out for 12 to 48 hours, and specifically, for example, the intensity can be 56mw/cm 2 12 to 48h (e.g., 12, 16, 20, 24, 28, 32, 36, 40, 44, or 48 h); and/or in step (7), the acetic acid solution of collagen contains collagen with a mass fraction of 4-8% (e.g. 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5% or 8%), preferably 5%; in the present invention, the acetic acid solution of collagen (abbreviated as collagen solution) is prepared by mixing an acetic acid solution and collagen, and preferably, the acetic acid solution contains acetic acid at a concentration (mass fraction) of 4 to 8%.
According to some preferred embodiments, the electrospinning parameters for preparing the upper and/or lower dermis layers are: a voltage of 15 to 25kV (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 kV), a receiving distance of 10 to 17cm (e.g., 10, 11, 12, 13, 14, 15, 16, or 17 cm), and a flow rate of the first spinning liquid and/or the second spinning liquid of 1 to 3mL/h (e.g., 1, 1.5, 2, 2.5, or 3 mL/h); the present invention found that not any electrospinning parameters can be used to produce the upper and lower dermis layers, and in some more preferred embodiments, in step (5), the mass ratio of collagen to silk fibroin is 2:1, the concentration of the first spinning solution is 6%, the concentration of the second spinning solution is 8%, and the electrospinning parameters for producing the upper and lower dermis layers are: the voltage is 20kV, the receiving distance is 11cm, and the flow rates of the first spinning solution and the second spinning solution are 2mL/h, so that the upper dermis layer and the lower dermis layer which have good spinning effect, high spinning efficiency, uniform spinning and smooth surfaces can be obtained; according to the invention, the first spinning solution and the second spinning solution with different concentrations are adopted, and the fiber diameters and the pore diameters of the obtained upper dermis layer and the lower dermis layer are adjusted under the appropriate electrospinning parameters, so that the problem of disordered porous structures of the existing artificial skin is effectively solved.
According to some preferred embodiments, the acetic acid solution contains acetic acid at a concentration (mass fraction) of 4 to 8% (e.g., 4%, 5%, 6%, 7%, or 8%), preferably 5%.
According to some preferred embodiments, in step (2), the stirring time is 2 to 4h (e.g. 2, 2.5, 3, 3.5 or 4 h); and/or in step (3), the stirring time is 1 to 3 hours (e.g., 1, 1.5, 2, 2.5, or 3 hours).
According to some preferred embodiments, the collagen is type I collagen (i.e. type I collagen).
According to some specific embodiments, the preparation of the drug-loaded artificial skin comprises the following steps:
step S1, silk fibroin preparation, which specifically comprises the following steps:
step S1-1: adding 0.1-0.5g of mulberry silk into 0.5 wt% of Na 2 CO 3 Boiling the solution for 30 minutes, repeating the boiling for three times, degumming, taking out the silk and drying the silk;
step S1-2: adding CaCl into the silk 2 /C 2 H 5 OH/H 2 Dissolving the O ternary solution at 65 + -5 deg.C for 2-4h, cooling at room temperature, centrifuging at 8000-10000r/min for 10-15 min, and collecting supernatant to obtain silk fibroin solution; in the CaCl 2 /C 2 H 5 OH/H 2 In ternary O solution, caCl 2 、C 2 H 5 OH and H 2 The molar ratio of O is 1;
step S1-3: filling the dissolved silk fibroin solution into a dialysis bag (the molecular weight of the dialysis bag is 8000-14000) for dialysis for 48-72h, wherein the dialysate is purified water, and the water is changed every 12 h;
step S1-4: freeze-drying the dialyzed silk fibroin to prepare regenerated silk fibroin, namely the silk fibroin preferred by the invention; the conditions for freeze drying for preparing the silk fibroin are not particularly limited, and the conventional freeze drying conditions can be adopted.
Step S2, preparing type I collagen, which specifically comprises the following steps:
step S2-1: removing fat and fascia from bovine Achilles tendon, cleaning, and freezing;
step S2-2: cutting the frozen bovine achilles tendon to a thickness of about 0.8 mm;
step S2-3: soaking the slices cut by the S2-2 in a sodium bicarbonate solution with the mass fraction of 1.0-1.5% for 12-24h, and then washing the slices with purified water for a plurality of times to degrease;
step S2-4: adding the degreased slices of S2-3 into an acid solution with pH of 1-3, performing enzymolysis for 48-96h at the temperature of 0-25 ℃, and removing terminal peptides 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 pH of 2 is preferably used for enzymolysis, because the optimal conditions for activating the added pepsin are normal temperature and pH value of 2-3, the normal temperature enzymolysis is carried out for 48-96h, preferably 96h, the enzymolysis is complete, and the yield is high;
step S2-5: centrifuging the enzymolysis liquid obtained in the step S2-4 by using a centrifugal machine, taking supernate, 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, for example, a supersaturated salt solution;
step S2-6: adding the white floccules obtained in the step S2-5 into a dialysis bag for dialysis, and in order to ensure that the environmental change of the external dialysis liquid is mild and avoid the change of the external dialysis liquid from being too severe so as to cause irreversible precipitation of collagen in the internal dialysis liquid, adopting a gradient dialysis mode, such as gradually reducing the concentration of acetic acid in the external liquid, and finally using purified water for dialysis to complete the purification of the collagen; the way of gradient dialysis may be, for example: firstly, putting a dialysis bag into 45L of dialysis solution with pH =3 for dialysis for 4 days, wherein the dialysis temperature is 15 +/-3 ℃, and the dialysis solution is replaced for 1 time every 2 days; then placing the dialysis bag in 45L of dialysate with pH =4, dialyzing for 5 days at 15 + -3 deg.C, and replacing the dialysate every 1 day for 1 time; dialyzing the dialysis bag in 45L purified water at 15 + -3 deg.C for 6 days, and changing the dialysate 3 times per day, once in the morning, at noon and at night; in the present invention, the pH =3 dialysate and the pH =4 dialysate are, for example, acetic acid solutions prepared from acetic acid and purified water, and the pH =3 dialysate may be prepared, for example, by: 45000mL of purified water is added into a dialysis tank, 146.694mL of acetic acid is accurately taken by a measuring cylinder and a pipette and added into the dialysis tank, and the mixture is uniformly stirred to obtain an acetic acid solution with pH = 3; the dialysate with pH =4 can be formulated, for example, as: 45000mL of purified water is added into a dialysis tank, 1.479mL of acetic acid is accurately taken by a pipette and added into the dialysis tank, and the mixture is stirred uniformly, so that the acetic acid solution with the pH =4 is obtained.
Step S2-7: placing the collagen gel with certain mass and solid content produced in the step S2-6 into a homogenizer, adding a certain mass of water to prepare a collagen solution with the concentration of 0.5-0.9%, wherein the homogenizing frequency is 20-50Hz, and the homogenizing time is 15-60min;
step S2-8: freeze-drying the homogenized collagen gel obtained in the step S2-7 to obtain I-type collagen with uniform texture; the freeze-drying conditions for preparing the type I collagen are not particularly limited, and conventional freeze-drying conditions can be adopted.
Step S3, preparing the epidermis layer of the drug-loaded artificial skin, which specifically comprises the following steps:
step S3-1: dissolving mupirocin with a certain mass in purified water to prepare a mupirocin solution with the mass fraction of 1%;
step S3-2: adding the prepared silk fibroin and the I-type collagen into an acetic acid solution with the mass fraction of 5% according to the mass ratio of 1/2-2/1 in sequence, and fully stirring for 2-4h to obtain a mixed solution of the silk fibroin and the collagen, namely a silk fibroin-collagen solution, wherein the concentration of the solution is 4% -7%;
step S3-3: mixing the mupirocin solution prepared in the step S3-1 and the silk fibroin-collagen solution prepared in the step S3-2, and stirring for 1-3 hours to obtain a mixed solution;
step S3-4: injecting the mixed solution prepared in the step S3-3 into a special polytetrafluoroethylene mold, wherein the bottom end of the mold is provided with a plurality of micropores (the aperture of each micropore is 60-110 microns) which are formed by laser, copper wires with the diameter of 50-100 microns and the length of 5cm are inserted into each micropore, a non-woven fabric net with the size of 140-300 meshes is inserted into each copper wire, and the non-woven fabric net is tiled at the bottom end of the mold; and injecting the mixed solution into the special polytetrafluoroethylene mold, and then performing pore forming by adopting a template method.
Step S3-5: directly drying the mold injected with the S3-4 at normal temperature;
step S3-6: demolding; in order to facilitate demoulding after drying, the surface of the copper wire is coated with glycerol before mould filling; demoulding to obtain a semitransparent film with the thickness of 0.07-1.2mm, wherein one surface of the film is a rough surface with a net structure;
step S3-7: the prepared film is subjected to ultraviolet irradiation for 12-48h, weak crosslinking is carried out, and a skin layer is prepared, wherein the rough surface of the skin layer has a net structure; the skin layer has a microporous structure formed by uniformly distributing a plurality of micropores, and preferably, the pore diameter of each micropore is 60-110 μm.
S4, preparing a dermis layer of the drug-loaded artificial skin, which specifically comprises the following steps:
s4-1: adding collagen and silk fibroin with certain mass into hexafluoroisopropanol solution or acetic acid solution according to the mass ratio of 1/2-2/1 to prepare first spinning solution with the concentration of 4% -6% and second spinning solution with the concentration of 7-9%;
s4-2: firstly, preparing a lower layer (corresponding to a lower corium layer) of the corium layer by using a second spinning solution, adjusting the parameters of electrospinning to ensure that the fiber diameter and the porosity of the lower layer of the corium layer are both high, and when a spinning film reaches 1-2mm, replacing the first spinning solution to ensure that the upper layer (corresponding to an upper corium layer) of the corium layer has thinner fibers and lower porosity, so as to prepare the film with gradient fiber diameter and porosity, wherein the total thickness of the film is 2-4mm, and the parameters of electrospinning for preparing the upper layer of the corium layer and/or the lower layer of the corium layer are as follows: a voltage of 15 to 25kV (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 kV), a receiving distance of 10 to 17cm (e.g., 10, 11, 12, 13, 14, 15, 16, or 17 cm), and a flow rate of the first spinning liquid and/or the second spinning liquid of 1 to 3mL/h (e.g., 1, 1.5, 2, 2.5, or 3 mL/h);
s4-3: and (3) carrying out ultraviolet irradiation on the film prepared by the S4-2 for 12-48h for crosslinking to prepare the dermis.
Step S5, preparing the drug-loaded artificial skin, which specifically comprises the following steps:
s5-1: coating a layer of 5% acetic acid solution of type I collagen with concentration on the rough surface of the epidermal layer prepared in the step S3, and covering the rough surface on the upper dermal layer of the S4-3 cross-linked dermal layer to obtain a composite film;
s5-2: and (5) carrying out freeze drying and crosslinking on the composite film obtained in the step (S5-1) to obtain the drug-loaded artificial skin.
In a third aspect, the invention provides a drug-loaded artificial skin prepared by the preparation method in the second aspect; the epidermal layer is formed by compounding collagen, silk fibroin and mupirocin; the upper dermis is a fine fiber structure layer consisting of collagen and silk fibroin, and the lower dermis is a coarse fiber structure layer consisting of collagen and silk fibroin; in the present invention, the fine fiber structure layer and the coarse fiber structure layer are relatively, preferably, the fine fiber structure layer is a fine fiber structure layer with a fiber diameter of 580-647nm and a porosity of 50-60%, and the coarse fiber structure layer is a coarse fiber structure layer with a fiber diameter of 700-780nm and a porosity of 65-80%.
According to some preferred embodiments, the drug-loaded skin has one or more of the following properties: the thickness of the surface layer is 0.07-1.2 mm; the skin layer has a micropore structure, the micropore structure is formed by uniformly distributing a plurality of micropores, and the aperture of each micropore is preferably 60-110 μm; the rough surface of the epidermal layer is provided with a net structure; the thickness of the corium layer is 2-4mm, and the thickness of the upper corium layer is 1-2 mm; the fiber diameter of the upper corium layer is 580-647nm, and the porosity is 50-60%; the fiber diameter of the lower corium layer is 700-780nm, and the porosity is 65-80%.
The invention will be further illustrated by way of example, but the scope of protection is not limited to these examples. The present invention is capable of other embodiments, and various changes and modifications may be made by one skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Before the following embodiments, a plurality of research experiments are performed on electrospinning parameters for preparing the upper dermis layer and the lower dermis layer, and corresponding electrospinning parameters and electrospinning results of the research experiments are shown in table 2 below; table 2 shows that in a plurality of research experiments carried out under the same spinning conditions of hexafluoroisopropanol as a solvent, a collagen-to-silk fibroin mass ratio of 2:1, an electrospinning solution concentration of 6%, a voltage of 20kV, a receiving distance of 11cm and a spinning solution flow rate of 2mL/h, the average diameters of nanofibers obtained under these same conditions are slightly different, but are distributed in the range of 580-647nm, and the spinning results are all good.
Figure BDA0003587311250000191
Figure BDA0003587311250000201
Example 1
Step S1, silk fibroin preparation, which specifically comprises the following steps:
step S1-1: adding 0.3g of mulberry silk into 0.5% of Na by mass 2 CO 3 Boiling in water for 30 min, repeating for three times, addingDegumming, taking out and drying the silk;
step S1-2: adding CaCl into the dried silk 2 /C 2 H 5 OH/H 2 Dissolving the ternary O solution at 65 +/-5 ℃ for 4h, cooling at room temperature at 9000r/min, centrifuging for 15min, and collecting supernatant to obtain silk fibroin solution; in the CaCl 2 /C 2 H 5 OH/H 2 In ternary O solution, caCl 2 、C 2 H 5 OH and H 2 The molar ratio of O is 1;
step S1-3: filling the dissolved silk fibroin solution into a dialysis bag (the molecular weight of the dialysis bag is 8000-14000) for dialysis for 72h, wherein the dialysate is purified water, and the water is changed every 12 h;
step S1-4: the dialyzed silk fibroin is freeze-dried to prepare regenerated silk fibroin, namely the silk fibroin adopted in the following steps.
Step S2, preparing type I collagen, which specifically comprises the following steps:
step S2-1: removing fat and fascia on bovine achilles tendon with a certain mass, cleaning, and freezing;
step S2-2: cutting the frozen bovine achilles tendon to a thickness of about 0.8 mm;
step S2-3: soaking the slices cut by the S2-2 in a sodium bicarbonate aqueous solution with the mass fraction of 1.5% for 24 hours, and then washing the slices with purified water for a plurality of times to degrease;
step S2-4: adding the degreased slices of S2-3 into an acetic acid solution with pH of 2, adding pepsin at normal temperature for enzymolysis for 96 hours, and removing terminal peptides to obtain an enzymolysis solution;
step S2-5: centrifuging the enzymolysis liquid obtained in the step S2-4 by using a centrifugal machine, taking supernate, and separating out collagen floccules by using a sodium chloride supersaturated solution;
step S2-6: adding the white floccule obtained in the step S2-5 into a dialysis bag for dialysis, firstly putting the dialysis bag into 45L of dialysis solution with pH =3 for dialysis for 4 days, wherein the dialysis temperature is 15 +/-3 ℃, and the dialysis solution is replaced for 1 time every 2 days; then placing the dialysis bag in 45L of dialysate with pH =4, dialyzing for 5 days at 15 + -3 deg.C, and replacing the dialysate every 1 day for 1 time; dialyzing the dialysis bag in 45L purified water at 15 + -3 deg.C for 6 days, and changing the dialysate 3 times per day, once in the morning, at noon and at night;
step S2-7: placing the collagen gel with certain mass and solid content produced in the step S2-6 in a homogenizer, adding a certain mass of water to prepare a collagen solution with the concentration (mass fraction) of 0.5%, wherein the homogenizing frequency is 40Hz, and the homogenizing time is 40min;
step S2-8: and (4) freeze-drying the homogenized collagen gel obtained in the step (S2-7) to obtain the I-type collagen with uniform texture.
Step S3, preparing the epidermis layer of the drug-loaded artificial skin, which specifically comprises the following steps:
step S3-1: dissolving mupirocin in purified water to obtain 1% mupirocin solution;
step S3-2: adding the prepared silk fibroin and I-type collagen into an acetic acid solution with the mass fraction of 5% according to the mass ratio of 1:2 in sequence, and fully stirring for 4 hours to obtain a mixed solution of the silk fibroin and the collagen, namely a silk fibroin-collagen solution, wherein the concentration of the silk fibroin-collagen solution is 4%;
step S3-3: mixing the mupirocin solution prepared in the step S3-1 and the silk fibroin-collagen solution prepared in the step S3-2, and stirring for 3 hours to obtain a mixed solution;
step S3-4: injecting the mixed solution prepared in the step S3-3 into a polytetrafluoroethylene mold, wherein the bottom end of the mold is provided with a plurality of micropores (the aperture of each micropore is 80 microns) which are formed by laser, copper wires with the diameter of 75 microns and the length of 5cm are inserted into each micropore, a 200-mesh nonwoven net is inserted into each copper wire, and the nonwoven net is laid on the bottom end of the mold; and (3) after the mixed solution is injected into the polytetrafluoroethylene mold, performing pore-forming by adopting a template method.
Step S3-5: directly drying the mold injected with the S3-4 for 15 hours at normal temperature;
step S3-6: demolding; in order to facilitate demoulding after drying, the surface of the copper wire is coated with glycerol before mould filling; obtaining a semitransparent film with the thickness of 1mm and a plurality of micropores with the pore diameter of 80 mu m after demoulding, wherein one surface of the film is a rough surface with a net structure;
step S3-7: the obtained film had a strength of 56mw/cm 2 And irradiating for 48 hours under ultraviolet light to carry out crosslinking to obtain the skin layer.
Step S4, preparing a dermis layer of the drug-loaded artificial skin, which specifically comprises the following steps:
s4-1: adding certain mass of type I collagen and silk fibroin into hexafluoroisopropanol according to the mass ratio of 2:1 to prepare a first spinning solution with the concentration of 6% and a second spinning solution with the concentration of 8%;
s4-2: firstly, preparing the lower layer of the corium layer by using a second spinning solution with the concentration of 8% through an electrospinning technology, so that the fiber diameter and the porosity of the lower layer of the corium layer are both high, when a spinning film reaches 1mm, replacing the first spinning solution with the concentration of 6%, so that the upper layer of the corium layer is thin and low in porosity, the film with the gradient fiber diameter and porosity is prepared, the total thickness of the film is 2mm, and the electrospinning parameters for preparing the upper layer of the corium layer and the lower layer of the corium layer are as follows: voltage 20kV, take-over distance 11cm, flow rates of the first and second spinning dope were both 2mL/h.
S4-3: the film prepared from S4-2 has the strength of 56mw/cm 2 Irradiating for 48 hours for crosslinking under ultraviolet light to obtain the dermis layer; the obtained dermis layers comprise an upper dermis layer with a fiber diameter (average diameter) of 598nm and a porosity of 56%, and a lower dermis layer with a fiber diameter (average diameter) of 740m and a porosity of 76%;
step S5, preparing the drug-loaded artificial skin, which specifically comprises the following steps:
s5-1: coating a layer of 5% acetic acid solution of type I collagen on the rough surface of the epidermal layer prepared in the step S3, and covering the rough surface on an upper dermal layer included in the dermal layer obtained by crosslinking the step S4-3 to obtain a composite film;
s5-2: the composite film obtained in S5-1 was subjected to freeze-drying (freeze-drying under the conditions shown in Table 1) and crosslinking (at a strength of 56 mw/cm) 2 Irradiating for 48 hours under ultraviolet light to obtain the drug-loaded artificial skin.
Example 2
Example 2 is essentially the same as example 1, except that:
in step S3-4: injecting the mixed solution prepared in the step S3-3 into a common polytetrafluoroethylene mold capable of forming a skin layer;
step S3-5: directly drying the mold injected with the S3-4 at normal temperature;
step S3-6: demolding to obtain a film with smooth surface and no micropore structure;
step S3-7: the obtained film had a strength of 56mw/cm 2 And irradiating for 48 hours under ultraviolet light to carry out crosslinking to obtain the skin layer.
Example 3
Example 3 is essentially the same as example 1, except that:
s4, preparing a dermis layer of the drug-loaded artificial skin, which specifically comprises the following steps:
s4-1: adding a certain mass of type I collagen and silk fibroin into hexafluoroisopropanol according to a mass ratio of 2:1 to prepare a spinning solution with the concentration of 6%;
s4-2: a film with the thickness of 2mm is prepared by an electrospinning technology by using a spinning solution with the concentration of 6%, and the electrospinning parameters for preparing the film are as follows: the voltage is 20kV, the receiving distance is 11cm, and the flow velocity of the spinning solution is 2mL/h;
s4-3: the film prepared from S4-2 has the strength of 56mw/cm 2 Irradiating for 48 hours under ultraviolet light to obtain a dermis layer; the dermis layer was made to have a fiber diameter (mean diameter) of 623nm and a porosity of 58%.
Example 4
Example 4 is essentially the same as example 1, except that:
s4, preparing a dermis layer of the drug-loaded artificial skin, which specifically comprises the following steps:
s4-1: adding a certain mass of type I collagen and silk fibroin into hexafluoroisopropanol according to a mass ratio of 2:1 to prepare a spinning solution with the concentration of 8%;
s4-2: preparing a film with the thickness of 2mm by using spinning solution with the concentration of 8% through an electrospinning technology, wherein the electrospinning parameters for preparing the film are as follows: the voltage is 20kV, the receiving distance is 11cm, and the flow rate of the first spinning solution and the second spinning solution is 2mL/h;
s4-3: carrying out ultraviolet irradiation on the film prepared by the step S4-2 for 48h for crosslinking to prepare a dermis layer; the dermis layer was prepared with a fiber diameter (mean diameter) of 715nm and a porosity of 72%.
Comparative example 1
An artificial skin added with growth factors was prepared by the same method as in example 1 of the chinese patent application CN 101716375B.
Comparative example 2
Comparative example 2 an artificial skin was prepared in the same manner as in example 1 of the chinese patent application CN106729985 a.
The invention tests the air permeability, tear strength and tensile strength of the artificial skin in examples 1-4 and comparative examples 1-2; wherein, the air permeability test is as follows: the determination is carried out by adopting a moisture permeable cup method, anhydrous CaCl is filled in a breathable cup 2 The artificial skin is packaged in a cup mouth and is placed in an environment with the temperature of 37 ℃ and the relative humidity of 80 percent, the weight is measured every 1h, and the calculation formula is as follows:
air permeability =24 × Δ m/s t
In the formula: delta m-two-times weighing difference of same sample assembly
S-area of sample
T-test time
6 samples were tested and averaged.
The tear strength test adopts a three-spring Mizhongshi SLD 1000Z-type tear strength tester and a GB/T16578.2-2009 standard method for testing; the tensile strength test adopts a XLW-PC type intelligent electronic tensile machine and a GB/T1040.3-2006 standard method to test, and the results are shown in Table 3.
Table 3: the results of the performance tests of air permeability, tear strength and tensile strength of the artificial skins in examples 1 to 4 and comparative examples 1 to 2.
Figure BDA0003587311250000251
The present invention also performed wound repair experiments on the artificial skin of examples 1 to 4 and comparative examples 1 to 2, and the results are shown in table 4.
The experimental method comprises the following steps: first, experimental mice were anesthetized. Secondly, removing the hair on the back of the mouse by using depilatory cream, and disinfecting with alcohol; 1.0cm multiplied by 1.0cm full-layer skin defect wound surfaces are manufactured on the back of a mouse, and the depth of each mouse wound surface is ensured to be basically the same. Third, the artificial skin grafts prepared in examples 1 to 4 and comparative examples 1 to 2 were covered on the wound surface, wound surface healing rates were calculated at 7 th and 14 th days after the artificial skin graft, the wound surface healing rates were ratios of wound surface healing areas to initial wound surface areas, the results are shown in table 4, and whether the wound surface was infected after the artificial skin graft was observed, and the results are shown in table 4.
Table 4: results of artificial skin wound repair in examples 1 to 4 and comparative example 2.
Figure BDA0003587311250000261
The invention has not been described in detail and is in part known to those of skill in the art.
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, and not to limit it; although the present 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 solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.

Claims (12)

1. A medicine-carrying type artificial skin is characterized in that:
the drug-loaded artificial skin comprises an epidermal layer and a dermal layer;
the epidermal layer is formed by compounding collagen, silk fibroin and mupirocin; the surface layer has a microporous structure, the microporous structure is formed by uniformly distributing a plurality of micropores, and the aperture of each micropore is 60-110 μm;
the dermis layers comprise an upper dermis layer and a lower dermis layer, the upper dermis layer is a fine fiber structure layer consisting of collagen and silk fibroin, and the lower dermis layer is a coarse fiber structure layer consisting of collagen and silk fibroin; the fiber diameter of the upper dermal layer is 580 to 647nm, and the porosity is 50 to 60 percent; the fiber diameter of the lower dermal layer is 700-780nm, and the porosity is 65-80%; the upper dermal layer is closer to the epidermal layer than the lower dermal layer.
2. The drug-loaded artificial skin of claim 1, wherein the drug-loaded artificial skin has one or more of the following properties:
the thickness of the epidermal layer is 0.07 to 1.2mm;
the thickness of the real leather layer is 2-4mm, and the thickness of the upper real leather layer is 1-2mm;
the surface of the epidermal layer, which is close to the upper dermal layer, is rough, the surface of the epidermal layer, which is far away from the upper dermal layer, is smooth, and the rough surface of the epidermal layer is provided with a reticular structure;
the upper dermis layer and the lower dermis layer are both prepared by an electrospinning technology.
3. The preparation method of the drug-loaded artificial skin is characterized in that the drug-loaded artificial skin comprises an epidermal layer and a dermal layer, the dermal layer comprises an upper dermal layer and a lower dermal layer, the epidermal layer has a microporous structure, the microporous structure is formed by uniformly distributing a plurality of micropores, and the pore diameter of each micropore is 60-110 mu m; the fiber diameter of the upper dermal layer is 580 to 647nm, and the porosity is 50 to 60 percent; the fiber diameter of the lower dermal layer is 700-780nm, and the porosity is 65-80%; the upper dermal layer is closer to the epidermal layer than the lower dermal layer;
the method comprises the following steps:
(1) Dissolving mupirocin in water to obtain mupirocin solution;
(2) Adding silk fibroin and collagen into an acetic acid solution and uniformly stirring to obtain a silk fibroin-collagen solution;
(3) Mixing the mupirocin solution and the silk fibroin-collagen solution and uniformly stirring to obtain a mixed solution;
(4) Injecting the mixed solution into a mold, carrying out pore-forming by a template method, and then drying, demolding and crosslinking to obtain a skin layer; the bottom end of the die is provided with a plurality of micropores which are formed by laser, copper wires with the diameter of 50-100 mu m are inserted into the micropores, a non-woven fabric net is inserted into the bottom of the copper wires, and the non-woven fabric net is laid on the bottom end of the die;
(5) Adding collagen and silk fibroin into hexafluoroisopropanol or acetic acid solution to prepare a first spinning solution with the mass concentration of 4~6% and a second spinning solution with the mass concentration of 7~9%; in the step (5), the mass ratio of the collagen to the silk fibroin is 2:1; the electrospinning parameters for preparing the upper and lower dermis layers were: the voltage is 15 to 25kV, the receiving distance is 10 to 17cm, and the flow rates of the first spinning solution and the second spinning solution are 1 to 3mL/h;
(6) Preparing a lower dermis layer by adopting the second spinning solution through an electrospinning technology, preparing an upper dermis layer on the basis of the lower dermis layer by adopting the first spinning solution through the electrospinning technology, and then preparing a dermis layer through crosslinking;
(7) Coating acetic acid solution of collagen on the epidermal layer, then covering the epidermal layer on the upper dermal layer of the dermal layer, and then carrying out freeze drying and crosslinking to prepare the drug-loaded artificial skin.
4. The production method according to claim 3, characterized in that:
one surface of the epidermal layer prepared in the step (4) is smooth, and the other surface of the epidermal layer is rough;
in the step (7), a collagen acetic acid solution is coated on the rough surface of the epidermis layer, and then the epidermis layer including the upper dermis layer is covered, and then the drug-loaded artificial skin is prepared by freeze drying and crosslinking.
5. The production method according to claim 3 or 4, characterized in that:
in the step (1), the mupirocin solution contains 0.5 to 1.5 mass percent of mupirocin;
in the step (2), the mass ratio of the silk fibroin to the collagen is 1: (0.5 to 2), wherein the mass concentration of the silk fibroin-collagen solution is 4~7%;
in the step (4), the non-woven fabric net is a non-woven fabric net with 140 to 300 meshes;
in the step (5), the mass concentration of the first spinning solution is 6%, and the mass concentration of the second spinning solution is 8%;
in step (4), step (6) and/or step (7), the crosslinking is: ultraviolet irradiation is carried out for 12 to 48h; and/or
In the step (7), the collagen acetic acid solution contains the collagen with the mass fraction of 4~8%.
6. The method of claim 5, wherein:
in the step (1), the mupirocin solution contains mupirocin in a mass fraction of 1%.
7. The method of claim 5, wherein:
in the step (7), the collagen acetic acid solution contains collagen in a mass fraction of 5%.
8. The production method according to claim 3, characterized in that:
the mass fraction of acetic acid contained in the acetic acid solution is 4~8%; and/or
The collagen is type I collagen.
9. The method of claim 8, wherein:
the mass fraction of acetic acid contained in the acetic acid solution is 5%.
10. The production method according to claim 3, characterized in that:
in the step (2), stirring for 2 to 4 hours; and/or
In the step (3), the stirring time is 1 to 3h.
11. The drug-loaded artificial skin prepared by the preparation method of any one of claims 3 to 10, which is characterized in that:
the epidermal layer is formed by compounding collagen, silk fibroin and mupirocin;
the upper dermis is a fine fiber structure layer composed of collagen and silk fibroin, and the lower dermis is a coarse fiber structure layer composed of collagen and silk fibroin.
12. The drug-loaded artificial skin of claim 11, wherein the drug-loaded skin has one or more of the following properties:
the thickness of the epidermal layer is 0.07 to 1.2mm;
one rough surface of the epidermal layer is provided with a net structure;
the thickness of the real leather layer is 2 to 4mm, and the thickness of the upper real leather layer is 1 to 2mm.
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