CN107715181A - A kind of preparation method of biodegradable organization engineering skin support - Google Patents

A kind of preparation method of biodegradable organization engineering skin support Download PDF

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Publication number
CN107715181A
CN107715181A CN201710802649.9A CN201710802649A CN107715181A CN 107715181 A CN107715181 A CN 107715181A CN 201710802649 A CN201710802649 A CN 201710802649A CN 107715181 A CN107715181 A CN 107715181A
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collagen
skin
human
organization engineering
skin support
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CN107715181B (en
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范代娣
朱晨辉
惠俊峰
米钰
郑晓燕
马沛
雷桓
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Northwest University
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Northwest University
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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
    • A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
    • A61L27/44—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
    • A61L27/48—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix with macromolecular fillers
    • 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
    • A61L27/58—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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J3/00—Processes of treating or compounding macromolecular substances
    • C08J3/24—Crosslinking, e.g. vulcanising, of macromolecules
    • C08J3/246—Intercrosslinking of at least two polymers
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J9/00—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof
    • C08J9/26—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof by elimination of a solid phase from a macromolecular composition or article, e.g. leaching out
    • 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
    • A61L2300/23—Carbohydrates
    • A61L2300/236—Glycosaminoglycans, e.g. heparin, hyaluronic acid, chondroitin
    • 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
    • A61L2300/252—Polypeptides, proteins, e.g. glycoproteins, lipoproteins, cytokines
    • 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
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2201/00—Foams characterised by the foaming process
    • C08J2201/04—Foams characterised by the foaming process characterised by the elimination of a liquid or solid component, e.g. precipitation, leaching out, evaporation
    • C08J2201/048—Elimination of a frozen liquid phase
    • C08J2201/0484—Elimination of a frozen liquid phase the liquid phase being aqueous
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2389/00—Characterised by the use of proteins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2405/00—Characterised by the use of polysaccharides or of their derivatives not provided for in groups C08J2401/00 or C08J2403/00
    • C08J2405/08—Chitin; Chondroitin sulfate; Hyaluronic acid; Derivatives thereof

Abstract

The invention discloses a kind of preparation method of biodegradable organization engineering skin support, auxiliary material carboxyl chitosan and Sodium Hyaluronate are added specially in the aqueous solution of human-like collagen, and add pore-foaming agent NaCl, after stirring is fully dissolved to all addition materials, after adding the progress enzyme process crosslinking of biology enzyme crosslinking agent glutamine transaminage, and sterilizing is irradiated by freezing, vacuum freeze drying, desalination and Co60, obtain degradable three-dimensional tissue's engineering skin support available for skin repair.The dermal scaffold has good biocompatibility and pore structure, and suitable cell increases, in the treatment available for full thickness dermal and burn and scald.

Description

A kind of preparation method of biodegradable organization engineering skin support
Technical field
The present invention relates to a kind of preparation method of biodegradable organization engineering skin support, belong to bio-medical material Technical field.
Background technology
Clinically burn and scald and defect of skin patient are badly in need of dermatoplasty, and auto-skin grafting, which has, does not occur immunological rejection The advantages of reaction, but for large area burned patient, its source is often definitely insufficient, and postoperative skin donor part may be deposited The slight illness such as self-heal, infection or cicatrization and risk are being unable to, therefore its application is subject to certain restrictions；Xenogenesis is of the same race Heterogenous skin transplanting has quantity abundance, relatively extensive etc. the advantage in source, but strong immune row can all occur after dermatoplasty Reprimand reaction, transplanting skin can often be fallen by rejection quickly, equally with a certain degree of limitation, it is therefore desirable to which one kind can substitute people The artificial skin of the skin of body in itself just arises at the historic moment.
Artificial skin refers to the principle and method of utilizing works and cell biology, the skin generation manually developed in vitro Articles for use, for repairing, substituting the skin histology of defect.This just needs to choose the good raw material of biocompatibility, then passes through Advanced technological means prepares organization engineering skin support, to meet cell adhesion, growth, migration, propagation and the load of differentiation Body, serve as " corium " part of organization engineering skin.
At present, the raw material of organization engineering skin support use the native biopolymers such as collagen, glycosaminoglycan more, such Material has good biocompatibility, suitable mechanics pliability, low immunogenicity and degradability.Though natural collagen is wide It is general to be recognized as a kind of most desirable material rich in skin, but presently used collagen is animal sources collagen egg In vain, it is big to there are sourcing limitations, composition mass uniformity difference caused by source animal difference uses acid with normal in preparation technology Chemical residual reagent toxicity caused by alkali, thereby increases and it is possible to which the not thorough problem that can be sterilized because of raw material causes rabid ox disease etc. animal derived Viral hidden danger.
Human-like collagen is a kind of water-soluble people source type human-like collagen prepared using modern advanced biotechnology, The unfavorable problem of animal sources collagen can not only be evaded completely, and it can effectively facilitate epithelial cell growth, promote defect The quick reparation of site tissue.
The content of the invention
It is an object of the invention to provide a kind of satisfactory mechanical property, the tissue work that biocompatibility is excellent, biodegradable The preparation method of journey dermal scaffold.
To achieve the above object, the technical solution adopted by the present invention is：
A kind of preparation method of biodegradable organization engineering skin support：Shell is added into human-like collagen solution to gather Sugar, Sodium Hyaluronate and pore-foaming agent NaCl, stir to all addition materials after fully dissolving, add biology enzyme crosslinking agent paddy ammonia Amide transaminase carry out enzyme process cross-linking reaction after, be transferred in mould freezed, superfreeze and vacuum freeze drying processing Afterwards, the dermal scaffold material of saliferous is obtained；Ultra-pure water washing by soaking is carried out to the dermal scaffold material of saliferous, removes inorganic salts and residual Monomer crosslinked dose stayed, then sterilizing is irradiated through secondary cryogenic freezing and vacuum freeze drying, and with Co60, it can be used for The biodegradable organization engineering skin support of skin repair.
Above-mentioned human-like collagen solution is that human-like collagen is dissolved in the mass volume ratio obtained in distilled water is dense Spend for 8-20%（g/ml）Human-like collagen solution.
The molecular weight of above-mentioned carboxyl chitosan is 15-20 ten thousand, and carboxylation degree is more than 60%, and viscosity is less than 30mPa s, and it is added Amount and the mass volume ratio concentration of solution are 0.5-4%（g/ml）.
Above-mentioned Sodium Hyaluronate molecular weight is 1,000,000, and the mass volume ratio concentration of its addition and solution is 0.05-1% （g/ml）.
For above-mentioned NaCl as perforating agent, the mass volume ratio concentration of its addition and solution is 0.5-3.5%（g/ml）.
Above-mentioned glutamine transaminage addition is 20-80U/g albumen, and static cross-linking reaction time is 8-24h, specifically Can be 8h, 12h, 18h or 24h etc.；Cross-linking reaction temperature is 4-45 DEG C, concretely 4 DEG C, 25 DEG C, 37 DEG C or 45 DEG C etc..
The above-mentioned dermal scaffold material to saliferous carries out ultra-pure water washing by soaking, soak time 2-4 days, is changed once per 8h Washings.
Sample need to carry out front and rear each 2-5h of refrigerator freezing, superfreeze, then vacuum twice after crosslinking of the present invention The Co60 radiation sterilizations of freeze-drying and the exposure dose for passing through >=25KGY can obtain available for skin repair can biology The organization engineering skin support of degraded.
Involved medicine and reagent can be obtained by commercially available approach in technical scheme.
The present invention is major phase material using human-like collagen, by adding Sodium Hyaluronate and carboxyl chitosan, and is selected Biology enzyme crosslinking agent glutamine transaminage is taken to prepare a kind of with good biocompatibility, rush epithelial cell growth, machinery Property good, inorganic body rejection and viral hidden danger novel degradable three-dimensional tissue engineering skin timbering material, fully meet complete The treatment of layer defect of skin and burn and scald needs, and major advantage is as follows：
（1）Human-like collagen involved in the present invention is the people source collagen type of genetic engineering bacterium high density fermentation, is had Excellent biocompatibility, promoting growth of cell, without animal virus hidden danger the characteristics of, have the work(of extraordinary wound healing Effect；
（2）There are active carboxyl and amino in carboxyl chitosan used in the present invention, they have stronger chemical reaction ability, Can strengthen and adjust acquisition organization engineering skin support mechanical strength and degradability, its elecrtonegativity is more beneficial for cell Stick.In addition chitosan, which has, suppresses bacterial activity, the promotion effect having had to wound healing and sterilizing and anti-virus；
（3）Hyaluronic acid used in the present invention is one of main matrix composition of application on human skin epidermis and corium, and its physiological function is Moisture can be made to enter space between cells, and with protein with reference to and form protein gel, cell is sticked together, that brings into normal play is thin Born of the same parents' metabolism, play and keep cell moisture, protect cell not encroached on by pathogen, accelerate to recover skin histology, improve wound Mouth healing power of regeneration, reduces the effect such as scar, strengthen immunity.The use of Sodium Hyaluronate undoubtedly adds organizational project skin The skin ultrastructure ability of skin support；
（4）Crosslinking agent glutamine transaminage used in the present invention is a kind of biological enzyme agent being present in human body, can be with The acry radical donor (γ-carboxamide groups in glutamine residue) and acyl acceptor of catalytic proteins（Amino）Between react life Into isopeptide bond, realize that intramolecular and intermolecular generation covalent cross-linking occur for protein and peptide.The use of the crosslinking agent is not only Effect to collagen molecules, additionally it is possible to synthetic cell epimatrix, influence cell propagation and cell chemotaxis, promote wound face Regeneration, therefore the remaining of monomer after cross-linking is without removing；
（5）Preparation method raw material of the present invention is easy to get, and cost is low, and synthesis technique is simply easily realized, product quality is stable, technique amplification Easily and reproduction performance is good.Novel degradable three-dimensional tissue engineering skin timbering material prepared by the present invention is in skins such as burn and scalds There is good application prospect in terms of skin defect repair.
Embodiment
With reference to embodiment, the present invention will be described in detail.
Embodiment 1：The preparation of scaffold materials of tissue-engineered skin
Step 1：Weigh human-like collagen to be dissolved in 10ml distilled waters, it is molten to obtain the human-like collagen that mass fraction is 10% Liquid, and carboxyl chitosan, Sodium Hyaluronate and perforating agent NaCl are added thereto, the mass volume ratio concentration of addition and solution 2%, 0.5% and 2.5% respectively is, stirring and dissolving is uniform；
Step 2：Biology enzyme crosslinking agent glutamine transaminage is added into the solution of step 1, addition is 60U/g albumen, It is to be mixed uniformly after be transferred to square dies, be placed in 4 DEG C of refrigerator stand crosslinking 24h；Refrigerator freezing is carried out again and -80 DEG C ultralow After warm each 3h of freezing processing, the dermal scaffold material of saliferous is obtained through vacuum freeze drying；
Step 3：The saliferous dermal scaffold material freezed in step 2 ultra-pure water washing by soaking 3 days, once washing is changed per 8h Water, after removing salinity and residual cross-linker, sample is placed in refrigerator freezing and each 3h of -80 DEG C of superfreeze again, and through true Vacuum freecing-dry and Co60 radiation sterilizations（>=25KGY exposure dose）, obtain final organization engineering skin support.
The microstructure Sample Scan electron microscope of the organization engineering skin support sample obtained in the present embodiment 1 is shown in it Portion's structure is porous network structure, even aperture distribution and pore size is between 30-500 μm, is adapted to cell growth；Experiment The swelling behavior of organization engineering skin support is have detected, it is swelled result in ultra-pure water and physiological saline and shows that support passes through 2h Afterwards with regard to that can reach swelling equilibrium, the water retention of different temperatures sub-mount material, which changes with time, shows that timbering material has relatively The ability of good locking moisture.
Mechanics Performance Testing is carried out to dermal scaffold material in the present embodiment 1, the results showed that the organization engineering skin branch The compression stress of frame increases with the increase of compression distance, and stress reaches 0.5MPa when compressing 0.8mm/mm, and tensile stress is maximum Up to 70kPa, it can meet the requirement of dermal scaffold material.
Embodiment 2：The preparation of scaffold materials of tissue-engineered skin
Step 1：Weigh human-like collagen to be dissolved in 10ml distilled waters, it is molten to obtain the human-like collagen that mass fraction is 20% Liquid, and carboxyl chitosan, Sodium Hyaluronate and perforating agent NaCl are added thereto, the mass volume ratio concentration of addition and solution 1%, 0.25% and 3% respectively is, stirring and dissolving is uniform；
Step 2：Biology enzyme crosslinking agent glutamine transaminage is added into the solution of step 1, addition is 50U/g albumen, It is to be mixed uniformly after be transferred to square dies, be placed in 25 DEG C of refrigerator stand crosslinking 18h；Refrigerator freezing is carried out again and -80 DEG C super After each 3h of cryogenic freezing processing, the dermal scaffold material of saliferous is obtained through vacuum freeze drying；
Step 3：The saliferous dermal scaffold material freezed in step 2 ultra-pure water washing by soaking 4 days, once washing is changed per 8h Water, after removing salinity and residual cross-linker, sample is placed in refrigerator freezing and each 3h of -80 DEG C of superfreeze again, and through true Vacuum freecing-dry and Co60 radiation sterilizations（>=25KGY exposure dose）, obtain final organization engineering skin support.
The organization engineering skin support of the scaffold materials of tissue-engineered skin of gained and gained in embodiment 1 in the embodiment Material physicochemical property is similar.
Embodiment 3：The preparation of scaffold materials of tissue-engineered skin
Step 1：Weigh human-like collagen to be dissolved in 10ml distilled waters, it is molten to obtain the human-like collagen that mass fraction is 15% Liquid, and carboxyl chitosan, Sodium Hyaluronate and perforating agent NaCl are added thereto, the mass volume ratio concentration of addition and solution 1.5%, 0.1% and 2.5% respectively is, stirring and dissolving is uniform；
Step 2：Biology enzyme crosslinking agent glutamine transaminage is added into the solution of step 1, addition is 20U/g albumen, It is to be mixed uniformly after be transferred to square dies, be placed in 37 DEG C of refrigerator stand crosslinking 12h；Refrigerator freezing is carried out again and -80 DEG C super After each 3h of cryogenic freezing processing, the dermal scaffold material of saliferous is obtained through vacuum freeze drying；
Step 3：The saliferous dermal scaffold material freezed in step 2 ultra-pure water washing by soaking 2 days, once washing is changed per 8h Water, after removing salinity and residual cross-linker, sample is placed in refrigerator freezing and each 3h of -80 DEG C of superfreeze again, and through true Vacuum freecing-dry and Co60 radiation sterilizations（>=25KGY exposure dose）, obtain final organization engineering skin support.
The organization engineering skin support of the scaffold materials of tissue-engineered skin of gained and gained in embodiment 1 in the embodiment Material physicochemical property is similar.
Embodiment 4：The preparation of scaffold materials of tissue-engineered skin
Step 1：Weigh human-like collagen to be dissolved in 10ml distilled waters, it is molten to obtain the human-like collagen that mass fraction is 8% Liquid, and carboxyl chitosan, Sodium Hyaluronate and perforating agent NaCl are added thereto, the mass volume ratio concentration of addition and solution 4%, 0.8% and 1% respectively is, stirring and dissolving is uniform；
Step 2：Biology enzyme crosslinking agent glutamine transaminage is added into the solution of step 1, addition is 40U/g albumen. It is to be mixed uniformly after be transferred to square dies, be placed in 45 DEG C of refrigerator stand crosslinking 8h；Refrigerator freezing is carried out again and -80 DEG C ultralow After warm each 3h of freezing processing, the dermal scaffold material of saliferous is obtained through vacuum freeze drying；
Step 3：The saliferous dermal scaffold material freezed in step 2 ultra-pure water washing by soaking 3 days, once washing is changed per 8h Water, after removing salinity and residual cross-linker, sample is placed in refrigerator freezing and each 3h of -80 DEG C of superfreeze again, and through true Vacuum freecing-dry and Co60 radiation sterilizations（>=25KGY exposure dose）, obtain final organization engineering skin support.
The organization engineering skin support of the scaffold materials of tissue-engineered skin of gained and gained in embodiment 1 in the embodiment Material physicochemical property is similar.
Embodiment 5：The cell toxicity test of scaffold materials of tissue-engineered skin
Examined using L929 cell CCK-8 methods and cytotoxicity is carried out to the scaffold materials of tissue-engineered skin prepared in embodiment 1 Detection.The leaching liquor that dermal scaffold material is soaked in obtained timbering material in simulated body fluid is used for L929 cell culture, cultivates After five days, cell survival rate is more than 160%, shows that the material cell growth has obvious facilitation.According to national standard, Material toxic grade is 0 grade, belongs to safe bio-medical material.
Embodiment 6：The application of scaffold materials of tissue-engineered skin
Experiment carries out full thickness dermal using New Zealand's experimental rabbit to the scaffold materials of tissue-engineered skin prepared by embodiment 1 Reparative experiment.The full thickness dermal reparative experiment at new zealand rabbit back shows the good histocompatbility of the timbering material, Being compared with control group, material can effectively stop dust and the pathogen of outside air, avoid tissue from suppurating, and wound is deepened, And the experimental group control group that compares can promote wound faster to heal.
Prepared scaffold materials of tissue-engineered skin carries out full thickness dermal reparation to new zealand rabbit in embodiment 1 Effect shows：H＆E coloration results show that at the 3rd day, epidermal structure and pore structure still lacked for experimental group and control group, wound Mouth is by cell, exudate and inflammatory mediator composition.Substantial amounts of inflammatory cell is produced by body, for antibacterial and repairs wound, this Outside, many granulation tissues are found in wound deep layer.When reaching the 7th day, over time, wound area is increasingly It is small.It is observed that some hair follicles outgrowths in regeneration zone, the epidermis at the top of regenerating tissues has formd, by contrast, Newborn mamilla on the animal wound of experimental group is more more ripe than control-animal, at the 14th day, the whole table of experimental group Skin is formed.There is no fresh corium exposure in atmosphere, cell and ECM density become similar to normal skin, and hypodermis is got over Come abundanter, for the skin of test group of animals, more hair follicles outgrowths occupy regenerating tissues.

Claims (7)

  1. A kind of 1. preparation method of biodegradable organization engineering skin support, it is characterised in that：It is molten to human-like collagen Chitosan, Sodium Hyaluronate and pore-foaming agent NaCl are added in liquid, stirs to all addition materials after fully dissolving, adds biology After enzyme crosslinking agent glutamine transaminage carries out enzyme process cross-linking reaction, be transferred in mould freezed, superfreeze and vacuum After freeze-drying process, the dermal scaffold material of saliferous is obtained；Ultra-pure water washing by soaking is carried out to the dermal scaffold material of saliferous, removed Remove inorganic salts and residual monomer crosslinked dose, then through secondary cryogenic freezing and vacuum freeze drying, and be irradiated and gone out with Co60 Bacterium, obtain the biodegradable organization engineering skin support available for skin repair.
  2. 2. according to the method for claim 1, it is characterised in that：Human-like collagen solution is to dissolve human-like collagen The mass volume ratio concentration obtained in distilled water is 8-20% collagen solution.
  3. 3. according to the method for claim 1, it is characterised in that：The molecular weight of carboxyl chitosan is 15-20 ten thousand, and carboxylation degree is big In 60%, viscosity is less than 30mPa s, and the mass volume ratio concentration of its addition and solution is 0.5-4%.
  4. 4. according to the method for claim 1, it is characterised in that：Sodium Hyaluronate molecular weight be 1,000,000, its addition with it is molten The mass volume ratio concentration of liquid is 0.05-1%.
  5. 5. according to the method for claim 1, it is characterised in that：NaCl is as perforating agent, the quality of its addition and solution Volume by volume concentration is 0.5-3.5%.
  6. 6. according to the method for claim 1, it is characterised in that：Glutamine transaminage addition is 20-80U/g albumen, Static cross-linking reaction time is 8-24h, and cross-linking reaction temperature is 4-45 DEG C.
  7. 7. according to the method for claim 1, it is characterised in that：After crosslinking sample carry out refrigerator freezing, superfreeze and Vacuum freeze drying, and the Co60 radiation sterilizations for the exposure dose for passing through >=25KGY obtain available for skin repair can biology The organization engineering skin support of degraded.
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CN111359006A (en) * 2020-03-22 2020-07-03 福建医科大学 Medical high-strength high-antibacterial-property enzymolysis-resistant gel membrane and preparation method thereof
CN112870453A (en) * 2020-07-07 2021-06-01 深圳市第二人民医院(深圳市转化医学研究院) Gelatin-type III collagen hydrogel and preparation method and application thereof
CN113750286A (en) * 2021-09-30 2021-12-07 振德医疗用品股份有限公司 Skin wound covering film and preparation method thereof
CN114258310A (en) * 2019-12-09 2022-03-29 爱德华兹生命科学公司 Bioprosthetic tissue preparation

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CN102363798A (en) * 2011-11-15 2012-02-29 无锡贝迪生物工程有限公司 Preparation process for collagen sponge

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CN1820790A (en) * 2006-03-13 2006-08-23 西北大学 Method for preparing biological degradable tissue engineering rack material
CN102363798A (en) * 2011-11-15 2012-02-29 无锡贝迪生物工程有限公司 Preparation process for collagen sponge

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Publication number Priority date Publication date Assignee Title
CN114258310A (en) * 2019-12-09 2022-03-29 爱德华兹生命科学公司 Bioprosthetic tissue preparation
CN111359006A (en) * 2020-03-22 2020-07-03 福建医科大学 Medical high-strength high-antibacterial-property enzymolysis-resistant gel membrane and preparation method thereof
CN111359006B (en) * 2020-03-22 2023-03-28 福建医科大学 Medical high-strength high-antibacterial-property enzymolysis-resistant gel film and preparation method thereof
CN112870453A (en) * 2020-07-07 2021-06-01 深圳市第二人民医院(深圳市转化医学研究院) Gelatin-type III collagen hydrogel and preparation method and application thereof
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