CN111803709A - Artificial skin and preparation method thereof - Google Patents
Artificial skin and preparation method thereof Download PDFInfo
- Publication number
- CN111803709A CN111803709A CN202010587419.7A CN202010587419A CN111803709A CN 111803709 A CN111803709 A CN 111803709A CN 202010587419 A CN202010587419 A CN 202010587419A CN 111803709 A CN111803709 A CN 111803709A
- Authority
- CN
- China
- Prior art keywords
- layer
- dermis
- solution
- artificial skin
- pore
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000002360 preparation method Methods 0.000 title claims abstract description 33
- 210000004207 dermis Anatomy 0.000 claims abstract description 206
- 210000003491 skin Anatomy 0.000 claims abstract description 116
- 239000004005 microsphere Substances 0.000 claims abstract description 53
- 239000000022 bacteriostatic agent Substances 0.000 claims abstract description 26
- 238000013268 sustained release Methods 0.000 claims abstract description 26
- 239000012730 sustained-release form Substances 0.000 claims abstract description 26
- 239000011148 porous material Substances 0.000 claims abstract description 18
- 210000002615 epidermis Anatomy 0.000 claims abstract description 14
- 239000011664 nicotinic acid Substances 0.000 claims abstract description 11
- 238000009826 distribution Methods 0.000 claims abstract description 10
- 108010022355 Fibroins Proteins 0.000 claims description 126
- 239000000243 solution Substances 0.000 claims description 115
- 230000002500 effect on skin Effects 0.000 claims description 83
- 229920002413 Polyhexanide Polymers 0.000 claims description 75
- 239000002245 particle Substances 0.000 claims description 73
- 239000011259 mixed solution Substances 0.000 claims description 72
- 239000003795 chemical substances by application Substances 0.000 claims description 66
- 102000008186 Collagen Human genes 0.000 claims description 58
- 108010035532 Collagen Proteins 0.000 claims description 58
- 229920001436 collagen Polymers 0.000 claims description 58
- 238000000034 method Methods 0.000 claims description 47
- 239000011159 matrix material Substances 0.000 claims description 29
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 28
- 238000004132 cross linking Methods 0.000 claims description 23
- 238000002156 mixing Methods 0.000 claims description 22
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 21
- 239000007788 liquid Substances 0.000 claims description 14
- 229910052757 nitrogen Inorganic materials 0.000 claims description 14
- 238000007873 sieving Methods 0.000 claims description 14
- 238000005507 spraying Methods 0.000 claims description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 14
- 238000004108 freeze drying Methods 0.000 claims description 13
- 239000012528 membrane Substances 0.000 claims description 10
- 238000001914 filtration Methods 0.000 claims description 6
- 238000010257 thawing Methods 0.000 claims description 6
- 238000005119 centrifugation Methods 0.000 claims description 5
- 239000002105 nanoparticle Substances 0.000 claims description 5
- 239000002077 nanosphere Substances 0.000 claims description 5
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 4
- 239000000741 silica gel Substances 0.000 claims description 4
- 229910002027 silica gel Inorganic materials 0.000 claims description 4
- 238000002604 ultrasonography Methods 0.000 claims description 3
- 230000029663 wound healing Effects 0.000 abstract description 15
- 230000001737 promoting effect Effects 0.000 abstract description 10
- 230000003385 bacteriostatic effect Effects 0.000 abstract description 9
- 230000035876 healing Effects 0.000 abstract description 5
- 238000007710 freezing Methods 0.000 description 14
- 230000008014 freezing Effects 0.000 description 14
- 230000000844 anti-bacterial effect Effects 0.000 description 12
- 230000000694 effects Effects 0.000 description 10
- 239000000725 suspension Substances 0.000 description 10
- 206010052428 Wound Diseases 0.000 description 8
- 208000027418 Wounds and injury Diseases 0.000 description 8
- 238000001035 drying Methods 0.000 description 8
- 239000000706 filtrate Substances 0.000 description 8
- 238000004140 cleaning Methods 0.000 description 7
- 238000002054 transplantation Methods 0.000 description 7
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 6
- 230000015556 catabolic process Effects 0.000 description 6
- 238000006731 degradation reaction Methods 0.000 description 6
- 208000015181 infectious disease Diseases 0.000 description 5
- 238000012986 modification Methods 0.000 description 5
- 230000004048 modification Effects 0.000 description 5
- 239000003242 anti bacterial agent Substances 0.000 description 4
- 230000009286 beneficial effect Effects 0.000 description 4
- 230000002349 favourable effect Effects 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 239000002994 raw material Substances 0.000 description 4
- 210000002950 fibroblast Anatomy 0.000 description 3
- 230000005012 migration Effects 0.000 description 3
- 238000013508 migration Methods 0.000 description 3
- 230000008929 regeneration Effects 0.000 description 3
- 238000011069 regeneration method Methods 0.000 description 3
- 238000001132 ultrasonic dispersion Methods 0.000 description 3
- 230000007547 defect Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 206010059866 Drug resistance Diseases 0.000 description 1
- 206010063560 Excessive granulation tissue Diseases 0.000 description 1
- 206010060872 Transplant failure Diseases 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 230000036770 blood supply Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 239000000645 desinfectant Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 210000001126 granulation tissue Anatomy 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 229920001296 polysiloxane Polymers 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000036573 scar formation Effects 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 238000005728 strengthening Methods 0.000 description 1
- 230000008467 tissue growth Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 230000037314 wound repair Effects 0.000 description 1
Classifications
-
- 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
- A61L27/26—Mixtures of macromolecular compounds
-
- 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
-
- 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
- A61L27/58—Materials at least partially resorbable by the body
-
- 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
- A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
- A61L2300/204—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials with nitrogen-containing functional groups, e.g. aminoxides, nitriles, guanidines
- A61L2300/206—Biguanides, e.g. chlorohexidine
-
- 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
-
- 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
- A61L2300/412—Tissue-regenerating or healing or proliferative agents
-
- 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
- A61L2300/604—Biodegradation
-
- 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
- A61L2300/62—Encapsulated active agents, e.g. emulsified droplets
- A61L2300/622—Microcapsules
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Transplantation (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Dermatology (AREA)
- Oral & Maxillofacial Surgery (AREA)
- Epidemiology (AREA)
- Biomedical Technology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Dispersion Chemistry (AREA)
- Cardiology (AREA)
- Heart & Thoracic Surgery (AREA)
- Vascular Medicine (AREA)
- Materials For Medical Uses (AREA)
Abstract
The invention belongs to the field of medical instruments, and particularly relates to an artificial skin and a preparation method thereof. The artificial skin comprises a dermis layer and an epidermis layer, and is characterized in that the dermis layer comprises an upper dermis layer and a lower dermis layer; wherein, the upper layer of the dermis layer comprises sustained-release microspheres loaded with bacteriostatic agents; the dermis is a bionic structure with pore size gradient distribution, and the pore size of the upper layer of the dermis is smaller than that of the lower layer of the dermis; the aperture of the upper layer of the dermis layer is 20-100 μm, and the aperture of the lower layer of the dermis layer is 110-200 μm. In the artificial skin provided by the invention, the sustained-release microspheres loaded with the bacteriostatic agent are introduced into the upper layer of the dermis layer, so that the artificial skin is endowed with good and continuous bacteriostatic activity, and the bionic structure with the dermis layer aperture gradient has more advantages in promoting wound healing of patients and improving healing quality.
Description
Technical Field
The invention belongs to the field of medical instruments, and particularly relates to an artificial skin and a preparation method thereof.
Background
The artificial skin can be used as substitute for autologous skin transplantation, and can be used for treating wounds with damaged dermal layer, chronic wound or burn wound. The artificial skin has the effects of promoting wound healing, reducing scar formation and the like, overcomes the defect of insufficient resources of autograft skin of a large-area burn patient, and gradually occupies the mainstream of the market. However, artificial skin fails to establish blood supply with wound surface in early stage after transplantation, so that the artificial skin has poor bacteriostatic ability and high infection rate after transplantation. Infection can not only directly result in graft failure, but can even threaten the life of the patient.
At present, the clinically common measures for preventing infection after artificial skin transplantation mainly comprise: thoroughly cleaning wound surface, soaking artificial skin in disinfectant before transplantation, strengthening dressing change after transplantation, using antibiotics for the whole body, and the like. Although the measures can play a certain role in preventing infection after transplantation, the artificial skin cannot be endowed with antibacterial capacity, and the organ of a patient is easily damaged by using a large amount of the medicine, so that the negative effect on the body of the patient is great.
In order to solve the problem of infection after clinical skin grafting, the method adopted in the prior art mainly comprises the following steps: adding bacteriostatic agent into the dermis of artificial skin, or introducing bacteriostatic layer between the dermis and epidermis of artificial skin. The artificial skin prepared by directly adding the bacteriostatic agent into the dermis has bacteriostatic ability but does not have a slow release function, and the overhigh bacteriostatic concentration in the early use stage can bring toxic effect to a wound surface and a human body to a certain degree; the antibacterial layer is introduced between the dermis layer and the epidermis layer, so that the problem of slow release of the antibacterial agent is solved, but the antibacterial layer is not a component of the artificial skin which plays a role in wound repair, and has defects and shortcomings in the aspects of wound vascularization and granulation tissue growth. In addition, the prior art mainly focuses on endowing the artificial skin with antibacterial and bacteriostatic properties, neglects the bionic structure and degradation property of the dermis, and has the problems of poor healing promotion effect and the like.
Therefore, there is a need for an artificial skin with sustained-release antibacterial activity and a preparation method thereof.
Disclosure of Invention
In view of the above problems, the present invention provides an artificial skin with sustained-release antibacterial activity and a preparation method thereof. In the preparation method, the sustained-release microspheres loaded with the bacteriostatic agent are introduced into the upper layer of the dermis layer, so that the artificial skin is endowed with good and continuous bacteriostatic activity; in addition, in the artificial skin provided by the invention, the dermis is a bionic structure with pore size gradient distribution, and has more advantages in promoting wound healing of patients and improving healing quality.
The technical scheme of the invention for realizing the purpose is as follows:
the invention provides an artificial skin, which comprises a dermis layer and an epidermis layer, and is characterized in that the dermis layer comprises an upper dermis layer and a lower dermis layer;
wherein, the upper layer of the dermis layer comprises sustained-release microspheres loaded with bacteriostatic agents;
the dermis is a bionic structure with pore size gradient distribution, and the pore size of the upper layer of the dermis is smaller than that of the lower layer of the dermis;
the aperture of the upper layer of the dermis layer is 20-100 μm, and the aperture of the lower layer of the dermis layer is 110-200 μm.
In some embodiments, in the artificial skin of the present invention, the sustained release microspheres are fibroin nanoparticles, and the bacteriostatic agent is PHMB (polyhexamethylene biguanide hydrochloride);
in some embodiments, in the artificial skin according to the present invention, the epidermal layer is a medical microporous silicone membrane.
In some embodiments, in the artificial skin of the present invention, the thickness of the upper layer of the dermal layer is 0.5 to 1.0 mm.
In some embodiments, the thickness of the lower layer of the dermal layer in the artificial skin of the invention is 2-4 mm.
The invention also provides a preparation method of the artificial skin, which comprises the following steps:
preparing sustained-release microspheres loaded with a bacteriostatic agent;
preparing a pore-forming agent at the upper layer of the dermis layer and a pore-forming agent at the lower layer of the dermis layer;
preparing a mixed solution of collagen and silk fibroin;
mixing the mixed solution (mixed solution of collagen and silk fibroin), the pore-forming agent on the upper layer of the dermis layer and the sustained-release microspheres loaded with the bacteriostatic agent to obtain a matrix solution on the upper layer of the dermis layer;
mixing the mixed solution (mixed solution of collagen and silk fibroin) with the dermal layer lower layer pore-forming agent to obtain a dermal layer lower layer matrix solution;
freeze-drying the upper matrix solution of the dermis layer to obtain the upper layer of the dermis layer;
freeze-drying the dermal layer lower layer matrix solution to obtain the dermal layer lower layer;
superposing the upper dermis layer and the lower dermis layer to obtain the dermis layer;
bonding the upper layer of the dermis layer with the epidermis layer to obtain the artificial skin;
wherein the particle size of the pore-forming agent on the upper layer of the dermis layer is 20-100 mu m; the particle size of the lower-layer pore-forming agent of the dermis layer is 110-200 mu m.
In some embodiments, in the method for preparing artificial skin, the bacteriostatic agent is PHMB, and the sustained-release microspheres are fibroin nanoparticles;
the preparation of the sustained-release microsphere loaded with the bacteriostatic agent comprises the following steps: preparing a silk fibroin solution and a PHMB solution, and carrying out freeze thawing, centrifugation, filtration, ultrasound and freeze drying on a mixed solution of the silk fibroin solution and the PHMB solution to obtain the PHMB-loaded silk fibroin nano-microspheres.
In some embodiments, the method for preparing artificial skin according to the present invention, the freeze-thawing of the mixed solution of the silk fibroin solution and the PHMB solution comprises: freezing the mixed solution of the silk fibroin solution and the PHMB solution at-60 to-50 ℃ for 10 to 12 hours, and then unfreezing at 18 to 28 ℃.
In some embodiments, the method for preparing the artificial skin according to the present invention, the preparing the PHMB-loaded silk fibroin nanospheres comprises: preparing a silk fibroin solution and a PHMB solution, and performing freeze thawing on a mixed solution of the silk fibroin solution and the PHMB solution to obtain a suspension of the PHMB-loaded silk fibroin nano-microspheres; and centrifuging, filtering, ultrasonically treating and freeze-drying the PHMB-loaded fibroin nano-microsphere suspension to obtain the PHMB-loaded fibroin nano-microsphere.
In some embodiments, in the method for preparing artificial skin of the present invention, the centrifugation comprises: centrifugally cleaning for 2-4 times at a rotating speed of 5500-6500 rpm.
In some embodiments, in the preparation method of the artificial skin, the concentration of the silk fibroin solution is 5.0-10.0 mg/mL; the concentration of the PHMB solution is 0.5-2.0 mg/mL;
in some embodiments, in the method for preparing artificial skin, in the mixed solution of the silk fibroin solution and the PHMB solution, the mass ratio of the silk fibroin to the PHMB is (80-120): 1.
In some embodiments, in the method for preparing artificial skin according to the present invention, the filtering process comprises: sequentially passing through filter membranes with the aperture of 0.3 mu m and the aperture of 0.1 mu m, and collecting filtrate with the particle size of 0.1-0.3 mu m.
In some embodiments, in the preparation method of the artificial skin, the particle size of the PHMB-loaded fibroin nanospheres is 100-300 nm.
In some embodiments, the method for preparing artificial skin according to the present invention, wherein the preparing the pore-forming agent for the upper layer of the dermal layer comprises: spraying pure water into liquid nitrogen to prepare ice particles, sieving the ice particles at low temperature, and collecting the ice particles with the particle size of 20-100 mu m to obtain a pore-forming agent on the upper layer of the dermis layer;
the preparation of the dermal layer lower layer pore-forming agent comprises the following steps: and spraying pure water into the liquid nitrogen to prepare ice particles, sieving the ice particles at low temperature, and collecting the ice particles with the particle size of 110-200 mu m to obtain the dermal layer lower layer pore-forming agent.
In some embodiments, the method for preparing artificial skin according to the present invention, wherein the preparing the pore-forming agent for the upper layer of the dermal layer comprises: spraying pure water into liquid nitrogen to prepare ice particles, sieving the ice particles at the temperature of-20 to-10 ℃, and collecting the ice particles with the particle size of 20 to 100 mu m to obtain a pore-forming agent on the upper layer of the dermis layer;
the preparation of the dermal layer lower layer pore-forming agent comprises the following steps: spraying pure water into liquid nitrogen to prepare ice particles, sieving the ice particles at the temperature of-20 to-10 ℃, and collecting the ice particles with the particle size of 110 to 200 mu m to obtain the lower-layer pore-forming agent of the dermis.
In some embodiments, in the method for preparing artificial skin according to the present invention, the preparing a mixed solution of collagen and silk fibroin includes: preparing a collagen solution with the concentration of 0.1-1.0 mg/mL and a silk fibroin solution with the concentration of 0.1-1.0 mg/mL, and mixing the collagen solution and the silk fibroin solution to obtain a mixed solution of the collagen and the silk fibroin.
In some embodiments, in the method for preparing artificial skin, the mass ratio of the collagen to the silk fibroin in the mixed solution of the collagen and the silk fibroin is (0.8-1.1): (0.9-1), preferably 0.9: 1.
In some embodiments, in the method for preparing artificial skin according to the present invention, the mixed solution (mixed solution of collagen and silk fibroin), the pore-forming agent on the upper layer of the dermal layer, and the sustained-release microspheres loaded with the bacteriostatic agent are mixed at a temperature of-10 to-4 ℃ to obtain a matrix solution on the upper layer of the dermal layer.
In some embodiments, the method for preparing artificial skin according to the present invention comprises mixing the mixed solution with the dermal layer lower layer pore-forming agent at a temperature of-10 to-4 ℃ to obtain a dermal layer lower layer matrix solution.
In some embodiments, the preparation method of the artificial skin of the present invention comprises placing the dermal layer upper matrix solution in a mold, freezing at-60 to-50 ℃ for 11 to 13 hours, and drying to obtain the dermal layer upper layer.
In some embodiments, the method for preparing artificial skin of the present invention comprises placing the dermal layer lower layer matrix solution in a mold, freezing at-60 to-50 ℃ for 11 to 13 hours, and drying to obtain the dermal layer lower layer.
In some embodiments, in the preparation method of the artificial skin, in the upper matrix solution of the dermis layer, the mass ratio of the mixed solution (the mixed solution of the collagen and the silk fibroin), the pore-forming agent of the upper layer of the dermis layer, and the antibacterial agent-loaded sustained-release microspheres is (9-11): 0.01-0.02);
in the basal body solution at the lower layer of the dermis, the mass ratio of the mixed solution (the mixed solution of the collagen and the silk fibroin) to the pore-forming agent at the lower layer of the dermis is (19-21): (19-21).
In some embodiments, the method for preparing artificial skin according to the present invention, wherein the overlaying the upper dermal layer and the lower dermal layer comprises: and sequentially adhering, crosslinking and analyzing the upper layer of the dermis layer and the lower layer of the dermis layer.
In some embodiments, the method for preparing artificial skin according to the present invention, the adhering the upper dermal layer and the lower dermal layer comprises: and reacting the upper layer of the dermis layer and the lower layer of the dermis layer for 2-5 hours under the conditions that the temperature is 37-52 ℃, the humidity is 70-80% and the concentration of acetic acid steam is 0.1-0.5%.
In some embodiments, the method for preparing artificial skin according to the present invention, the adhering the upper dermal layer and the lower dermal layer comprises: and superposing the upper layer of the dermis layer and the lower layer of the dermis layer according to the aperture distribution rule of the dermis layer of human skin, and then reacting for 2-5 hours under the conditions that the temperature is 37-52 ℃, the humidity is 70-80%, and the concentration of acetic acid vapor is 0.1-0.5%.
In some embodiments, the method for preparing artificial skin according to the present invention, the cross-linking the upper dermal layer and the lower dermal layer comprises: and (3) crosslinking the upper layer of the dermis layer and the lower layer of the dermis layer for 2-5 hours at the temperature of 37-52 ℃, the humidity of 70-80% and the concentration of glutaraldehyde steam of 10-30%, and then crosslinking for 12-48 hours at the temperature of 105-108 ℃ and the vacuum degree of 80-100 Pa.
In some embodiments, the method for preparing artificial skin according to the present invention, wherein analyzing the upper dermal layer and the lower dermal layer comprises: and analyzing the upper layer of the dermis layer and the lower layer of the dermis layer at the temperature of 37-52 ℃ for 2-5 h.
The invention also provides the artificial skin prepared by the preparation method of the artificial skin.
One or more technical embodiments of the present invention have at least the following technical effects or advantages:
(1) in the artificial skin provided by the invention, the PHMB-loaded silk fibroin nano slow-release microspheres are introduced into the upper layer of the dermis layer, so that the artificial skin is endowed with good and continuous antibacterial activity.
(2) In the artificial skin provided by the invention, the dermis layer with a specific bionic structure is formed due to the aperture gradient of the upper layer of the dermis layer and the lower layer of the dermis layer. The lower layer of the dermis has large aperture, which is beneficial to the adhesion, migration and regeneration of fibroblasts, thereby accelerating vascularization and promoting wound healing; the pore diameter of the upper layer of the dermis is small, and the good anti-cicatrization effect is exerted at the later stage of wound healing.
(3) In the preparation method of the artificial skin, the obtained dermis takes the collagen and the silk fibroin as main raw materials, so that the dermis is endowed with better degradation performance and is beneficial to promoting wound healing.
(4) In the preparation method of the artificial skin, the porous structure with a specific aperture range can be prepared by introducing the dermal layer upper pore-forming agent and the dermal layer lower pore-forming agent with different particle sizes.
Detailed Description
The present invention will be described in detail below with reference to specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly apparent therefrom. It will be understood by those skilled in the art that these specific embodiments and examples are for the purpose of illustrating the invention and are not to be construed as limiting the invention.
Throughout the specification, unless otherwise specifically noted, terms used herein should be understood as having meanings as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is a conflict, the present specification will control.
Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be prepared by existing methods.
In order to solve the technical problems, the embodiment of the invention provides the following general ideas:
the invention provides an artificial skin, which comprises a dermis layer and an epidermis layer, and is characterized in that the dermis layer comprises an upper dermis layer and a lower dermis layer;
wherein, the upper layer of the dermis layer comprises sustained-release microspheres loaded with bacteriostatic agents;
the dermis is a bionic structure with pore size gradient distribution, and the pore size of the upper layer of the dermis is smaller than that of the lower layer of the dermis;
the aperture of the upper layer of the dermis layer is 20-100 μm, and the aperture of the lower layer of the dermis layer is 110-200 μm.
The design idea of the technical scheme is as follows: in the artificial skin, the bionic structure is formed due to the aperture gradient of the upper layer of the dermis layer and the lower layer of the dermis layer. The lower layer of the dermis has large aperture, which is beneficial to the adhesion, migration and regeneration of fibroblasts, thereby accelerating vascularization and promoting wound healing; the pore diameter of the upper layer of the dermis is small, and the good anti-cicatrization effect is exerted at the later stage of wound healing.
In some embodiments, in the artificial skin of the present invention, the sustained release microspheres are fibroin nanoparticles, and the bacteriostatic agent is PHMB.
The invention isThe PHMB-loaded silk fibroin nano slow-release microspheres are introduced into the upper layer of the dermis layer of the artificial skin, so that the artificial skin is endowed with good and continuous bacteriostatic activity. The inventor researches and discovers that MIC of PHMBDilution ofAnd MBCDilution ofMinimum, and the percentage of strains with reduced sensitivity is lowest; the inventor also finds that only low-dose PHMB can play an effective antibacterial function and effectively reduce the risk of harm to the human body; in addition, the PHMB has the lowest sensitivity reduction percentage to common infected strains, has good antibacterial activity to most strains, and is not easy to generate drug resistance.
In some embodiments, in the artificial skin of the present invention, the thickness of the upper layer of the dermal layer is 0.5 to 1.0 mm.
In some embodiments, the thickness of the lower layer of the dermal layer in the artificial skin of the invention is 2-4 mm.
The invention also provides a preparation method of the artificial skin, which comprises the following steps:
preparing sustained-release microspheres loaded with a bacteriostatic agent;
preparing a pore-forming agent at the upper layer of the dermis layer and a pore-forming agent at the lower layer of the dermis layer;
preparing a mixed solution of collagen and silk fibroin;
mixing the mixed solution (mixed solution of collagen and silk fibroin), the pore-forming agent on the upper layer of the dermis layer and the sustained-release microspheres loaded with the bacteriostatic agent to obtain a matrix solution on the upper layer of the dermis layer;
mixing the mixed solution (mixed solution of collagen and silk fibroin) with the dermal layer lower layer pore-forming agent to obtain a dermal layer lower layer matrix solution;
freeze-drying the upper matrix solution of the dermis layer to obtain the upper layer of the dermis layer;
freeze-drying the dermal layer lower layer matrix solution to obtain the dermal layer lower layer;
superposing the upper dermis layer and the lower dermis layer to obtain the dermis layer;
bonding the upper layer of the dermal layer to the epidermal layer of claim 1 or 2 to obtain the artificial skin;
wherein the particle size of the pore-forming agent on the upper layer of the dermis layer is 20-100 mu m; the particle size of the lower-layer pore-forming agent of the dermis layer is 110-200 mu m.
The design idea of the technical scheme is as follows: in the preparation method of the artificial skin, the porous structure with a specific aperture range can be prepared by introducing the dermal layer upper pore-forming agent and the dermal layer lower pore-forming agent with different particle sizes. In addition, the preparation method of the invention uses collagen and silk fibroin as main raw materials to prepare the dermis, which endows the dermis with better degradation performance and is beneficial to promoting the healing of wound surfaces.
In some embodiments, in the method for preparing artificial skin, the bacteriostatic agent is PHMB, and the sustained-release microspheres are fibroin nanoparticles;
the preparation of the sustained-release microsphere loaded with the bacteriostatic agent comprises the following steps: preparing a silk fibroin solution and a PHMB solution, and carrying out freeze thawing, centrifugation, filtration, ultrasound and freeze drying on a mixed solution of the silk fibroin solution and the PHMB solution to obtain the PHMB-loaded silk fibroin nano-microspheres.
The preparation method of the invention adopts the PHMB-loaded fibroin nano slow-release microspheres, which can endow the artificial skin with good and continuous antibacterial activity and has obvious healing promoting effect.
In some embodiments, the method for preparing artificial skin according to the present invention, the freeze-thawing of the mixed solution of the silk fibroin solution and the PHMB solution comprises: freezing the mixed solution of the silk fibroin solution and the PHMB solution at-60 to-50 ℃ for 10 to 12 hours, and then unfreezing at 18 to 31 ℃. The inventor limits that the particle size of the thawed microsphere suspension can be more concentrated by freezing for 10-12 hours at-60 to-50 ℃.
In some embodiments, in the method for preparing artificial skin of the present invention, the centrifugation comprises: centrifugally cleaning for 2-4 times at a rotating speed of 5500-6500 rpm. The centrifugal separation can be achieved by limiting the rotating speed to 5500-6500 rpm, the structure of microspheres in suspension can be influenced by excessively high rotating speed, and the separation and cleaning effect cannot be achieved by excessively low rotating speed.
In some embodiments, in the preparation method of the artificial skin, the concentration of the silk fibroin solution is 5.0-10.0 mg/mL; the concentration of the PHMB solution is 0.5-2.0 mg/mL. The inventor limits the concentration of the silk fibroin solution to be 5.0-10.0 mg/mL and the concentration of the PHMB solution to be 0.5-2.0 mg/mL, and the PHMB can be fully mixed after being dropped into the silk fibroin solution, so that the artificial skin is endowed with good and continuous antibacterial activity.
In some embodiments, in the method for preparing artificial skin, in the mixed solution of the silk fibroin solution and the PHMB solution, the mass ratio of the silk fibroin to the PHMB is (80-120): 1. The mass ratio of the silk fibroin to the PHMB is (80-120): 1, the loading rate is high under the condition, more effective microspheres can be obtained, and therefore the artificial skin is endowed with good and continuous antibacterial activity.
In some embodiments, in the method for preparing artificial skin according to the present invention, the filtering process comprises: sequentially passing through filter membranes with the aperture of 0.3 mu m and the aperture of 0.1 mu m, and collecting filtrate with the particle size of 0.1-0.3 mu m. When the pore size is too large, the PHMB is released too slowly; and when the pore diameter is too small, PHMB is released too fast, which is not favorable for promoting wound healing.
In some embodiments, in the preparation method of the artificial skin, the particle size of the PHMB-loaded fibroin nanospheres is 100-300 nm. The PHMB is released too slowly due to the microspheres with too large particle sizes; the microspheres with too small particle size release PHMB too quickly, which is not favorable for promoting wound healing.
In some embodiments, the method for preparing artificial skin according to the present invention, wherein the preparing the pore-forming agent for the upper layer of the dermal layer comprises: spraying pure water into the liquid nitrogen to prepare ice particles with the particle size of 20-50 microns; sieving the ice particles at low temperature to obtain a pore-forming agent on the upper layer of the dermis layer;
the preparation of the dermal layer lower layer pore-forming agent comprises the following steps: spraying pure water into the liquid nitrogen to prepare ice particles with the particle size of 100-120 mu m; and sieving the ice particles at low temperature to obtain the dermal layer lower layer pore-forming agent.
In some embodiments, in the method for preparing artificial skin according to the present invention, the preparing a mixed solution of collagen and silk fibroin includes: preparing a collagen solution with the concentration of 2.5-5 mu mol/L and a silk fibroin solution with the concentration of 2.5-5 mu mol/L, and mixing the collagen solution and the silk fibroin solution to obtain a mixed solution of the collagen and the silk fibroin;
in some embodiments, in the method for preparing artificial skin, the mass ratio of the collagen to the silk fibroin in the mixed solution of the collagen and the silk fibroin is (0.8-1.1): (0.9-1), preferably 0.9: 1. Under the condition of the proportion, the collagen and the fibroin are assembled together, which is favorable for forming a space bionic structure.
In some embodiments, in the preparation method of the artificial skin, in the upper matrix solution of the dermis layer, the mass ratio of the mixed solution (the mixed solution of the collagen and the silk fibroin), the pore-forming agent of the upper layer of the dermis layer, and the antibacterial agent-loaded sustained-release microspheres is (9-11): 0.01-0.02); the inventor discovers through screening tests that the mixed solution, the pore-forming agent on the upper layer of the dermis layer and the sustained-release microspheres loaded with the bacteriostatic agent are mixed according to the mass ratio, so that the better degradation performance of the dermis layer is optimally endowed, and the wound healing is promoted.
In the basal body solution at the lower layer of the dermis, the mass ratio of the mixed solution (the mixed solution of the collagen and the silk fibroin) to the pore-forming agent at the lower layer of the dermis is (19-21): (19-21). The inventor discovers through screening tests that the mixed solution and the pore-forming agent at the lower layer of the dermis layer are mixed according to the mass ratio, so that the better degradation performance of the dermis layer is optimally endowed, and the wound healing is promoted.
In some embodiments, the method for preparing artificial skin according to the present invention, wherein the overlaying the upper dermal layer and the lower dermal layer comprises: and sequentially adhering, crosslinking and analyzing the upper layer of the dermis layer and the lower layer of the dermis layer.
In some embodiments, the method for preparing artificial skin according to the present invention, the adhering the upper dermal layer and the lower dermal layer comprises: and reacting the upper layer of the dermis layer and the lower layer of the dermis layer for 2-5 hours under the conditions that the temperature is 37-52 ℃, the humidity is 70-80% and the concentration of acetic acid steam is 0.1-0.5%. The technical scheme is favorable for realizing seamless adhesion of the upper layer and the lower layer of the dermis, protects the space structure of the upper layer and the lower layer of the dermis to a great extent, and plays a role in activating subsequent glutaraldehyde steam crosslinking.
In some embodiments, the method for preparing artificial skin according to the present invention, the crosslinking the upper dermal layer and the lower dermal layer, and in some embodiments, the crosslinking the upper dermal layer and the lower dermal layer comprises: and (3) crosslinking the upper layer of the dermis layer and the lower layer of the dermis layer for 2-5 hours at the temperature of 37-52 ℃, the humidity of 70-80% and the concentration of glutaraldehyde steam of 10-30%, and then crosslinking for 12-48 hours at the temperature of 105-108 ℃ and the vacuum degree of 80-100 Pa.
In some embodiments, the method for preparing artificial skin according to the present invention, wherein analyzing the upper dermal layer and the lower dermal layer comprises: and analyzing the upper layer of the dermis layer and the lower layer of the dermis layer at the temperature of 37-52 ℃ for 2-5 h.
The artificial skin and the method for preparing the same according to the present application will be described in detail with reference to examples.
Example 1
The preparation method of the artificial skin specifically comprises the following steps:
(1) preparing the PHMB-loaded fibroin nano-microspheres:
preparing 5mg/mL silk fibroin solution and 0.5mg/mL PHMB solution, slowly dripping the PHMB solution into the silk fibroin solution, fully and uniformly mixing, freezing at-60 ℃ for 12h, and unfreezing at room temperature to obtain PHMB-loaded silk fibroin microsphere suspension; centrifuging and cleaning the suspension for 2 times at 5500rpm, then sequentially passing through filter membranes with the pore diameter of 0.3 mu m and 0.1 mu m, collecting filtrate components with the interception amount of 0.1-0.3 mu m, carrying out ultrasonic dispersion on the filtrate components, and carrying out freeze drying to obtain the PHMB-loaded fibroin nano-microspheres with the particle size of 100-300 nm;
wherein, in the mixed solution of the silk fibroin solution and the PHMB solution, the mass ratio of the silk fibroin to the PHMB is 80: 1.
(2) Preparing a pore-forming agent at the upper layer of the dermis layer and a pore-forming agent at the lower layer of the dermis layer:
spraying pure water into liquid nitrogen to prepare ice particles, sieving the obtained ice particles at the temperature of-10 ℃, and collecting the ice particles with the particle size of 20-100 mu m to obtain a pore-forming agent on the upper layer of the dermis;
spraying pure water into liquid nitrogen to prepare ice particles, sieving the obtained ice particles at-10 ℃, and collecting the ice particles with the particle size of 110-200 mu m to obtain a lower-layer pore-forming agent of the dermis layer;
(3) preparing a mixed solution of collagen and silk fibroin:
preparing a collagen solution with the concentration of 0.1mg/mL and a silk fibroin solution with the concentration of 0.1mg/mL, and mixing the collagen solution and the silk fibroin solution to obtain a mixed solution of collagen and silk fibroin;
wherein, in the mixed solution of the collagen and the silk fibroin, the mass ratio of the collagen to the silk fibroin is 0.8: 0.9.
(4) Mixing 9g of mixed solution (the mixed solution of the collagen and the silk fibroin), 9g of pore-forming agent on the upper layer of the dermis layer and 0.01g of PHMB-loaded silk fibroin nano microspheres at-4 ℃ to obtain a matrix solution on the upper layer of the dermis layer;
(5) mixing 19g of mixed solution (mixed solution of collagen and silk fibroin) with 21g of dermal layer lower layer pore-forming agent at-4 deg.C to obtain dermal layer lower layer matrix solution.
(6) Placing the upper matrix solution of the dermal layer obtained in step (4) in a 316L mould with a thickness of 115 × 95mm, freezing at-60 deg.C for 11h, and drying to obtain an upper layer of the dermal layer with a thickness of 0.5 mm.
(7) Placing the dermal layer lower layer matrix solution obtained in step (5) in a 316L mould with the thickness of 115 x 95mm, freezing at-60 ℃ for 11h, and drying to obtain the dermal layer lower layer with the thickness of 2 mm.
(8) Sequentially adhering, crosslinking and analyzing the upper layer of the dermis layer obtained in the step (6) and the lower layer of the dermis layer obtained in the step (7) to obtain the dermis layer;
wherein, the bonding includes: superposing the upper layer of the dermis layer and the lower layer of the dermis layer according to the aperture distribution rule of the dermis layer of human skin, and then reacting for 2 hours under the conditions that the temperature is 37 ℃, the humidity is 70% and the concentration of acetic acid vapor is 0.1%;
the crosslinking comprises: and (2) crosslinking the upper layer of the dermis layer and the lower layer of the dermis layer for 2 hours at the temperature of 37 ℃, the humidity of 70 percent and the concentration of glutaraldehyde steam of 10 percent, and then crosslinking for 12 hours at the temperature of 105 ℃ and the vacuum degree of 80 Pa.
The analysis comprises the following steps: the upper and lower dermis layers were resolved at 37 ℃ for 2 h.
(9) And (3) adhering the upper layer of the dermis layer in the dermis layer obtained in the step (8) with the medical microporous silica gel membrane of the epidermis layer to obtain the artificial skin.
Example 2:
the preparation method of the artificial skin specifically comprises the following steps:
(1) preparing the PHMB-loaded fibroin nano-microspheres:
preparing a silk fibroin solution of 8mg/mL and a PHMB solution of 0.8mg/mL, slowly dripping the PHMB solution into the silk fibroin solution, fully and uniformly mixing, freezing at-60 ℃ for 12h, and unfreezing at room temperature to obtain a PHMB-loaded silk fibroin microsphere suspension; centrifuging and cleaning the suspension for 2 times at 5500rpm, then sequentially passing through filter membranes with the pore diameter of 0.3 mu m and 0.1 mu m, collecting filtrate components with the interception amount of 0.1-0.3 mu m, carrying out ultrasonic dispersion on the filtrate components, and carrying out freeze drying to obtain the PHMB-loaded fibroin nano-microspheres with the particle size of 100-300 nm;
wherein, in the mixed solution of the silk fibroin solution and the PHMB solution, the mass ratio of the silk fibroin to the PHMB is 100: 1.
(2) Preparing a pore-forming agent at the upper layer of the dermis layer and a pore-forming agent at the lower layer of the dermis layer:
spraying pure water into liquid nitrogen to prepare ice particles, sieving the obtained ice particles at the temperature of-10 ℃, and collecting the ice particles with the particle size of 20-100 mu m to obtain a pore-forming agent on the upper layer of the dermis;
spraying pure water into liquid nitrogen to prepare ice particles, sieving the obtained ice particles at-10 ℃, and collecting the ice particles with the particle size of 110-200 mu m to obtain a lower-layer pore-forming agent of the dermis layer;
(3) preparing a mixed solution of collagen and silk fibroin:
preparing a collagen solution with the concentration of 1.0mg/mL and a silk fibroin solution with the concentration of 1.0mg/mL, and mixing the collagen solution and the silk fibroin solution to obtain a mixed solution of collagen and silk fibroin;
wherein, in the mixed solution of the collagen and the silk fibroin, the mass ratio of the collagen to the silk fibroin is 0.9: 1.
(4) Mixing 10g of mixed solution (the mixed solution of the collagen and the silk fibroin), 10g of dermal layer upper pore-forming agent and 0.01g of PHMB-loaded fibroin nano-microspheres at-8 ℃ to obtain dermal layer upper matrix solution;
(5) mixing 21g of mixed solution (mixed solution of collagen and silk fibroin) with 19g of dermal layer lower layer pore-forming agent at-8 deg.C to obtain dermal layer lower layer matrix solution.
(6) Placing the upper matrix solution of the dermal layer obtained in step (4) in a 316L mould with a thickness of 115 × 95mm, freezing at-60 deg.C for 11h, and drying to obtain an upper layer of the dermal layer with a thickness of 0.6 mm.
(7) Placing the dermal layer lower layer matrix solution obtained in step (5) in a 316L mould with the thickness of 115 x 95mm, freezing at-60 ℃ for 11h, and drying to obtain the dermal layer lower layer with the thickness of 2.2 mm.
(8) Sequentially adhering, crosslinking and analyzing the upper layer of the dermis layer obtained in the step (6) and the lower layer of the dermis layer obtained in the step (7) to obtain the dermis layer;
wherein, the bonding includes: superposing the upper layer of the dermis layer and the lower layer of the dermis layer according to the aperture distribution rule of the dermis layer of human skin, and then reacting for 5 hours under the conditions that the temperature is 52 ℃, the humidity is 80% and the concentration of acetic acid vapor is 0.5%;
the crosslinking comprises: and (2) crosslinking the upper layer of the dermis layer and the lower layer of the dermis layer for 5 hours at the temperature of 52 ℃, the humidity of 80% and the concentration of glutaraldehyde steam of 30%, and then crosslinking for 48 hours at the temperature of 108 ℃ and the vacuum degree of 100 Pa.
The analysis comprises the following steps: the upper and lower dermis layers were resolved at 37 ℃ for 4 h.
(9) And (3) adhering the upper layer of the dermis layer in the dermis layer obtained in the step (8) with the medical microporous silica gel membrane of the epidermis layer to obtain the artificial skin.
Example 3
The preparation method of the artificial skin specifically comprises the following steps:
(1) preparing the PHMB-loaded fibroin nano-microspheres:
preparing a 10mg/mL silk fibroin solution and a 2.0mg/mL PHMB solution, slowly dripping the PHMB solution into the silk fibroin solution, fully and uniformly mixing, freezing at-60 ℃ for 12h, and unfreezing at room temperature to obtain a PHMB-loaded silk fibroin microsphere suspension; centrifuging and cleaning the suspension for 2 times at 5500rpm, then sequentially passing through filter membranes with the pore diameter of 0.3 mu m and 0.1 mu m, collecting filtrate components with the interception amount of 0.1-0.3 mu m, carrying out ultrasonic dispersion on the filtrate components, and carrying out freeze drying to obtain the PHMB-loaded fibroin nano-microspheres with the particle size of 100-300 nm;
wherein, in the mixed solution of the silk fibroin solution and the PHMB solution, the mass ratio of the silk fibroin to the PHMB is 120: 1.
(2) Preparing a pore-forming agent at the upper layer of the dermis layer and a pore-forming agent at the lower layer of the dermis layer:
spraying pure water into liquid nitrogen to prepare ice particles, sieving the obtained ice particles at the temperature of minus 20 ℃, and collecting the ice particles with the particle size of 20-100 mu m to obtain a pore-forming agent on the upper layer of the dermis;
spraying pure water into liquid nitrogen to prepare ice particles, sieving the obtained ice particles at the temperature of-20 ℃, and collecting the ice particles with the particle size of 110-200 mu m to obtain a lower-layer pore-forming agent of the dermis layer;
(3) preparing a mixed solution of collagen and silk fibroin:
preparing a collagen solution with the concentration of 0.5mg/mL and a silk fibroin solution with the concentration of 0.8mg/mL, and mixing the collagen solution and the silk fibroin solution to obtain a mixed solution of collagen and silk fibroin;
wherein, in the mixed solution of the collagen and the silk fibroin, the mass ratio of the collagen to the silk fibroin is 1.0: 0.9.
(4) Mixing 10g of mixed solution (the mixed solution of the collagen and the silk fibroin), 10g of dermal layer upper pore-forming agent and 0.02g of PHMB-loaded fibroin nano-microspheres at-10 ℃ to obtain dermal layer upper matrix solution;
(5) mixing 20g of mixed solution (mixed solution of collagen and silk fibroin) with 20g of dermal layer lower layer pore-forming agent at-10 deg.C to obtain dermal layer lower layer matrix solution.
(6) Placing the upper matrix solution of the dermal layer obtained in step (4) in a 316L mould with a thickness of 115 × 95mm, freezing at-60 deg.C for 11h, and drying to obtain an upper layer of the dermal layer with a thickness of 0.6 mm.
(7) Placing the dermal layer lower layer matrix solution obtained in step (5) in a 316L mould with the thickness of 115 x 95mm, freezing at-60 ℃ for 11h, and drying to obtain the dermal layer lower layer with the thickness of 2.2 mm.
(8) Sequentially adhering, crosslinking and analyzing the upper layer of the dermis layer obtained in the step (6) and the lower layer of the dermis layer obtained in the step (7) to obtain the dermis layer;
wherein, the bonding includes: superposing the upper layer of the dermis layer and the lower layer of the dermis layer according to the aperture distribution rule of the dermis layer of human skin, and then reacting for 4 hours under the conditions that the temperature is 45 ℃, the humidity is 70% and the concentration of acetic acid vapor is 0.4%;
the crosslinking comprises: and (2) crosslinking the upper layer of the dermis layer and the lower layer of the dermis layer for 4 hours at the temperature of 45 ℃, the humidity of 75% and the concentration of glutaraldehyde steam of 25%, and then crosslinking for 25 hours at the temperature of 1.6 ℃ and the vacuum degree of 90 Pa.
The analysis comprises the following steps: the upper and lower dermis layers were resolved at 37 ℃ for 5 h.
(9) And (3) adhering the upper layer of the dermis layer in the dermis layer obtained in the step (8) with the medical microporous silica gel membrane of the epidermis layer to obtain the artificial skin.
One or more technical implementation schemes in embodiments 1-3 of the present invention prove that the present invention has at least the following technical effects or advantages:
(1) the silk fibroin nano slow-release microspheres loaded with PHMB are introduced into the upper layer of the dermis layer of the artificial skin, so that the artificial skin is endowed with good and continuous bacteriostatic activity.
(2) The dermis layer of the artificial skin comprises an upper dermis layer and a lower dermis layer, the dermis layer is of a bionic structure with pore size gradient distribution, the pore size of the lower dermis layer is larger and is 110-200 mu m, and adhesion, migration and regeneration of fibroblasts are facilitated, so that vascularization is accelerated, and wound healing is promoted; the aperture of the upper layer of the dermis is smaller and is 20-100 mu m, and the dermis plays a good anti-scarring effect at the later stage of wound healing (3) in the preparation method of the artificial skin, the obtained dermis takes collagen and silk fibroin as main raw materials, so that the dermis is endowed with better degradation performance, and the wound healing is facilitated.
Finally, it should also be noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Claims (10)
1. An artificial skin comprising a dermis layer and an epidermis layer, wherein the dermis layer comprises an upper dermis layer and a lower dermis layer;
wherein, the upper layer of the dermis layer comprises sustained-release microspheres loaded with bacteriostatic agents;
the dermis is a bionic structure with pore size gradient distribution, and the pore size of the upper layer of the dermis is smaller than that of the lower layer of the dermis;
the aperture of the upper layer of the dermis layer is 20-100 μm, and the aperture of the lower layer of the dermis layer is 110-200 μm.
2. The artificial skin according to claim 1, wherein the slow release microspheres are fibroin nanospheres, and the bacteriostatic agent is PHMB;
preferably, the epidermis layer is a medical microporous silica gel membrane.
3. The method of preparing artificial skin according to claim 1 or 2, comprising the steps of:
preparing sustained-release microspheres loaded with a bacteriostatic agent;
preparing a pore-forming agent at the upper layer of the dermis layer and a pore-forming agent at the lower layer of the dermis layer;
preparing a mixed solution of collagen and silk fibroin;
mixing the mixed solution, the pore-forming agent on the upper layer of the dermis layer and the sustained-release microspheres loaded with the bacteriostatic agent to obtain a basal solution on the upper layer of the dermis layer;
mixing the mixed solution with the lower-layer pore-forming agent of the dermis layer to obtain a lower-layer matrix solution of the dermis layer;
freeze-drying the upper matrix solution of the dermis layer to obtain the upper layer of the dermis layer;
freeze-drying the dermal layer lower layer matrix solution to obtain the dermal layer lower layer;
superposing the upper dermis layer and the lower dermis layer to obtain the dermis layer;
bonding the upper layer of the dermal layer to the epidermal layer of claim 1 or 2 to obtain the artificial skin;
wherein the particle size of the pore-forming agent on the upper layer of the dermis layer is 20-100 mu m; the particle size of the lower-layer pore-forming agent of the dermis layer is 110-200 mu m.
4. The method for preparing artificial skin according to claim 3, wherein the bacteriostatic agent is PHMB, and the slow-release microspheres are fibroin nanospheres;
the preparation of the sustained-release microsphere loaded with the bacteriostatic agent comprises the following steps: preparing a silk fibroin solution and a PHMB solution, and carrying out freeze thawing, centrifugation, filtration, ultrasound and freeze drying on a mixed solution of the silk fibroin solution and the PHMB solution to obtain PHMB-loaded silk fibroin nano-microspheres;
preferably, the concentration of the silk fibroin solution is 5.0-10.0 mg/mL; the concentration of the PHMB solution is 0.5-2.0 mg/mL;
preferably, in the mixed solution of the silk fibroin solution and the PHMB solution, the mass ratio of the silk fibroin to the PHMB is (80-120): 1.
5. The method for preparing artificial skin according to claim 4, wherein the PHMB-loaded fibroin nanoparticles have a particle size of 100-300 nm.
6. The method for preparing artificial skin according to claim 3 or 4, wherein the preparing the pore-forming agent on the upper layer of the dermis layer comprises: spraying pure water into liquid nitrogen to prepare ice particles, sieving the ice particles at low temperature, and collecting the ice particles with the particle size of 20-100 mu m to obtain a pore-forming agent on the upper layer of the dermis layer;
the preparation of the dermal layer lower layer pore-forming agent comprises the following steps: and spraying pure water into the liquid nitrogen to prepare ice particles, sieving the ice particles at low temperature, and collecting the ice particles with the particle size of 110-200 mu m to obtain the dermal layer lower layer pore-forming agent.
7. The method for preparing artificial skin according to claim 3 or 4, wherein the preparing of the mixed solution of collagen and silk fibroin comprises: preparing a collagen solution with the concentration of 0.1-1.0 mg/mL and a silk fibroin solution with the concentration of 0.1-1.0 mg/mL, and mixing the collagen solution and the silk fibroin solution to obtain a mixed solution of the collagen and the silk fibroin;
preferably, in the mixed solution of the collagen and the silk fibroin, the mass ratio of the collagen to the silk fibroin is (0.8-1.1): (0.9-1), preferably 0.9: 1.
8. The method for preparing artificial skin according to claim 3 or 4, wherein the mass ratio of the mixed solution, the pore-forming agent on the upper layer of the dermis layer and the sustained-release microspheres loaded with the bacteriostatic agent in the base solution on the upper layer of the dermis layer is (9-11): 0.01-0.02; in the base solution at the lower layer of the dermis, the mass ratio of the mixed solution to the pore-forming agent at the lower layer of the dermis is (19-21): (19-21).
9. The method for preparing artificial skin according to claim 3 or 4, wherein the overlaying the upper dermal layer and the lower dermal layer comprises: sequentially adhering, crosslinking and analyzing the upper layer of the dermis layer and the lower layer of the dermis layer;
preferably, the adhering the upper dermis layer and the lower dermis layer comprises: and reacting the upper layer of the dermis layer and the lower layer of the dermis layer for 2-5 hours under the conditions that the temperature is 37-52 ℃, the humidity is 70-80% and the concentration of acetic acid steam is 0.1-0.5%.
10. The artificial skin prepared by the method for preparing artificial skin according to any one of claims 4 to 9.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010587419.7A CN111803709A (en) | 2020-06-24 | 2020-06-24 | Artificial skin and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010587419.7A CN111803709A (en) | 2020-06-24 | 2020-06-24 | Artificial skin and preparation method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111803709A true CN111803709A (en) | 2020-10-23 |
Family
ID=72854987
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010587419.7A Pending CN111803709A (en) | 2020-06-24 | 2020-06-24 | Artificial skin and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111803709A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112716919A (en) * | 2021-01-21 | 2021-04-30 | 巍太医疗科技(常州)有限公司 | Skin protective agent and preparation method thereof |
| CN113577398A (en) * | 2021-07-08 | 2021-11-02 | 湖北中部医疗科技有限公司 | 3D printing artificial skin and preparation method thereof |
| CN114288474A (en) * | 2021-12-27 | 2022-04-08 | 湖北中部医疗科技有限公司 | Dermal layer and artificial skin for promoting hair follicle regeneration and preparation method thereof |
| CN114732954A (en) * | 2022-04-08 | 2022-07-12 | 奥精医疗科技股份有限公司 | Medicine-carrying type artificial skin and preparation method thereof |
| CN117629468A (en) * | 2023-11-03 | 2024-03-01 | 广东工业大学 | A multi-layer microstructure flexible pressure sensor with gradient structure and its preparation method and application |
Citations (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040126405A1 (en) * | 2002-12-30 | 2004-07-01 | Scimed Life Systems, Inc. | Engineered scaffolds for promoting growth of cells |
| CN103239758A (en) * | 2012-02-14 | 2013-08-14 | 深圳兰度生物材料有限公司 | Artificial dermis bracket and preparation method of artificial dermis bracket |
| EP2694124A1 (en) * | 2011-04-05 | 2014-02-12 | Universitätsklinikum Freiburg | Biocompatible and biodegradable gradient layer system for regenerative medicine and for tissue support |
| CN104068945A (en) * | 2014-06-27 | 2014-10-01 | 深圳齐康医疗器械有限公司 | Artificial skin and preparation method thereof |
| CN104984407A (en) * | 2015-07-01 | 2015-10-21 | 世科志扬（北京）医疗科技有限公司 | Tissue engineering artificial skin and preparation method thereof |
| CN105903082A (en) * | 2016-06-17 | 2016-08-31 | 三峡大学 | Biodegradable medical nano-composite porous material, preparing method and application thereof |
| CN106729985A (en) * | 2016-12-27 | 2017-05-31 | 广东泰宝医疗器械技术研究院有限公司 | A kind of long-acting promoting healing artificial skin and preparation method thereof |
| CN107456608A (en) * | 2017-07-27 | 2017-12-12 | 中山大学附属第医院 | Artificial skin with three-dimensional porous structure and preparation method thereof |
| CN108404209A (en) * | 2018-03-19 | 2018-08-17 | 武汉亚洲生物材料有限公司 | A kind of collagen-fibroin assembles sponge material, altogether assembling artificial skin and preparation method thereof altogether |
| CN108853590A (en) * | 2018-08-22 | 2018-11-23 | 上海长海医院 | A kind of artificial dermis |
| CN109602958A (en) * | 2019-01-11 | 2019-04-12 | 湖北中部医疗科技有限公司 | A kind of artificial skin and preparation method thereof |
| CN110025830A (en) * | 2019-04-29 | 2019-07-19 | 振德医疗用品股份有限公司 | A kind of sustained release antibacterial type artificial dermis model and its construction method |
| KR20200031018A (en) * | 2018-09-13 | 2020-03-23 | 한국과학기술연구원 | Epidermis and dermis matrix similar to the real skin and artificial skin comprising the same |
-
2020
- 2020-06-24 CN CN202010587419.7A patent/CN111803709A/en active Pending
Patent Citations (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040126405A1 (en) * | 2002-12-30 | 2004-07-01 | Scimed Life Systems, Inc. | Engineered scaffolds for promoting growth of cells |
| EP2694124A1 (en) * | 2011-04-05 | 2014-02-12 | Universitätsklinikum Freiburg | Biocompatible and biodegradable gradient layer system for regenerative medicine and for tissue support |
| CN103239758A (en) * | 2012-02-14 | 2013-08-14 | 深圳兰度生物材料有限公司 | Artificial dermis bracket and preparation method of artificial dermis bracket |
| CN104068945A (en) * | 2014-06-27 | 2014-10-01 | 深圳齐康医疗器械有限公司 | Artificial skin and preparation method thereof |
| CN104984407A (en) * | 2015-07-01 | 2015-10-21 | 世科志扬（北京）医疗科技有限公司 | Tissue engineering artificial skin and preparation method thereof |
| CN105903082A (en) * | 2016-06-17 | 2016-08-31 | 三峡大学 | Biodegradable medical nano-composite porous material, preparing method and application thereof |
| CN106729985A (en) * | 2016-12-27 | 2017-05-31 | 广东泰宝医疗器械技术研究院有限公司 | A kind of long-acting promoting healing artificial skin and preparation method thereof |
| CN107456608A (en) * | 2017-07-27 | 2017-12-12 | 中山大学附属第医院 | Artificial skin with three-dimensional porous structure and preparation method thereof |
| CN108404209A (en) * | 2018-03-19 | 2018-08-17 | 武汉亚洲生物材料有限公司 | A kind of collagen-fibroin assembles sponge material, altogether assembling artificial skin and preparation method thereof altogether |
| CN108853590A (en) * | 2018-08-22 | 2018-11-23 | 上海长海医院 | A kind of artificial dermis |
| KR20200031018A (en) * | 2018-09-13 | 2020-03-23 | 한국과학기술연구원 | Epidermis and dermis matrix similar to the real skin and artificial skin comprising the same |
| CN109602958A (en) * | 2019-01-11 | 2019-04-12 | 湖北中部医疗科技有限公司 | A kind of artificial skin and preparation method thereof |
| CN110025830A (en) * | 2019-04-29 | 2019-07-19 | 振德医疗用品股份有限公司 | A kind of sustained release antibacterial type artificial dermis model and its construction method |
Non-Patent Citations (3)
| Title |
|---|
| KAZUYA MATSUDA ET AL: ""Evaluation of a bilayer artificial skin capable of sustained release of an antibiotic"", 《BIOMATERIALS》 * |
| WANG FENG ET AL: ""Collagen/chitosan based two-compartment and bi-functional dermal scaffolds for skin regeneration"", 《MATERIALS SCIENCE & ENGINEERING C-MATERIALS FOR BIOLOGICAL APPLICATIONS》 * |
| 金剑: ""PHMB修饰的人工真皮的抗菌能力检测"", 《中国优秀博硕士学位论文全文数据库（硕士） 基础科学辑》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112716919A (en) * | 2021-01-21 | 2021-04-30 | 巍太医疗科技(常州)有限公司 | Skin protective agent and preparation method thereof |
| CN113577398A (en) * | 2021-07-08 | 2021-11-02 | 湖北中部医疗科技有限公司 | 3D printing artificial skin and preparation method thereof |
| CN114288474A (en) * | 2021-12-27 | 2022-04-08 | 湖北中部医疗科技有限公司 | Dermal layer and artificial skin for promoting hair follicle regeneration and preparation method thereof |
| CN114732954A (en) * | 2022-04-08 | 2022-07-12 | 奥精医疗科技股份有限公司 | Medicine-carrying type artificial skin and preparation method thereof |
| CN114732954B (en) * | 2022-04-08 | 2022-12-06 | 奥精医疗科技股份有限公司 | Medicine-carrying type artificial skin and preparation method thereof |
| CN117629468A (en) * | 2023-11-03 | 2024-03-01 | 广东工业大学 | A multi-layer microstructure flexible pressure sensor with gradient structure and its preparation method and application |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111803709A (en) | Artificial skin and preparation method thereof | |
| Priya et al. | Skin tissue engineering for tissue repair and regeneration | |
| US8865199B2 (en) | Biomatrix composition and methods of biomatrix seeding | |
| CN103007345B (en) | Antibacterial biological activity stent and preparation method thereof | |
| CN111714706B (en) | Vascular stent capable of promoting vascular cell proliferation and secreting extracellular matrix, preparation method of vascular stent and active artificial blood vessel | |
| US6699287B2 (en) | Dermal scaffold using alkaline pre-treated chitosan matrix or alkaline pre-treated chitosan and alkaline pre-treated collagen mixed matrix | |
| US9421229B2 (en) | Neural scaffolds | |
| JP2008504921A (en) | Cell-independent production of tissue equivalents | |
| US20090143287A1 (en) | Tissue regeneration substrate | |
| CN110772669A (en) | A bioink for 3D printing artificial skin | |
| JP2015532845A (en) | New scaffold for cardiac repair patches | |
| JP4847129B2 (en) | Wound healing promoter | |
| CN111437441A (en) | Drug-loaded KGN (KGN) nano-fiber scaffold and preparation method and application thereof | |
| CN114350162B (en) | Gradient pore structure silk fibroin film and preparation method thereof | |
| CN108261557B (en) | A kind of nanofiber membrane for wound healing and its preparation method and application | |
| JP5540301B2 (en) | Porous substrate, method for producing the same, and method of using the porous substrate | |
| KR101507589B1 (en) | Method for fabricating composite bone hemostatic material composed of chitosan hydrogel and electrospun gelatin/BCP | |
| CN118806992A (en) | A composite patch for rotator cuff repair and preparation method thereof | |
| KR101182417B1 (en) | Artificial Nanofiber Amnion Membranes and Method of Making The Same | |
| CN102114272A (en) | Method for preparing quaternized chitosan and plasmid DNA compound particle loaded skin regeneration material | |
| CN116370691A (en) | Hydrogel film containing nano cell vesicles and application of hydrogel film in diabetic wound repair dressing | |
| CN102671243A (en) | Quaternized chitosan/siRNA composite particle-loaded skin regeneration material and preparation method thereof | |
| JP2000245450A (en) | Sheet for cultured skin improving taking rate | |
| CN116196473B (en) | Three-dimensional porous permeable peripheral nerve sleeve with precisely controllable degradation period and preparation method thereof | |
| CN116763977B (en) | Dressing doped with rubidium-containing mesoporous bioactive glass loaded with AIE and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20201023 |