CN109125799A - GelMA hydrogel people takes off the preparation method of the three-dimensional double-deck auxiliary material of cell amnion - Google Patents
GelMA hydrogel people takes off the preparation method of the three-dimensional double-deck auxiliary material of cell amnion Download PDFInfo
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- CN109125799A CN109125799A CN201811029545.XA CN201811029545A CN109125799A CN 109125799 A CN109125799 A CN 109125799A CN 201811029545 A CN201811029545 A CN 201811029545A CN 109125799 A CN109125799 A CN 109125799A
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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
- A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
- A61L26/0061—Use of materials characterised by their function or physical properties
- A61L26/0085—Porous materials, e.g. foams or sponges
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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
- A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
- A61L26/0057—Ingredients of undetermined constitution or reaction products thereof
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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
- A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
- A61L26/0061—Use of materials characterised by their function or physical properties
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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
- A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
- A61L26/0061—Use of materials characterised by their function or physical properties
- A61L26/0066—Medicaments; Biocides
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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
- 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
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Abstract
The present invention relates to medical bio bressing fields, specifically, it is a kind of preparation method of three-dimensional double-deck auxiliary material of the de- cell amnion of GelMA hydrogel people, this method is a kind of method that electrostatic spinning joint photo-crosslinking technology prepares the three-dimensional double-deck dressing of the de- cell amnion of GelMA hydrogel/people, specific production method are as follows: gelatin forms methacryl amido gelatin (GelMA) by methacrylic anhydride (MA) graft modification, the GelMA of formation is passed through into electrostatic spinning technique, people is covered in porous fibrous to take off on cell amnion (D-HAM), it is prepared into three-dimensional Double sided biological active dressing, photoinitiator Irgacure2959 row photo-crosslinking is added in the dressing;By the dressing freeze-drying after photo-crosslinking, in γ irradiation sterilization, encapsulated under aseptic condition.The present invention has good anti-inflammatory, anti-infective and biocompatibility, can promote mucous membrane of mouth quick healing of cut.
Description
Technical field
The present invention relates to medical bio bressing fields, specifically, being that a kind of de- cell amnion of GelMA hydrogel people is three-dimensional
The preparation method of the double-deck auxiliary material.
Background technique
Mucous membrane of mouth position is relatively special, it covers oral surfaces, and front is connected with skin of lip, rear and throat mucous membrane
It is connected, the secretion etc. for entering to invade saliva by resisting mechanical irritation, limiting toxicity substance, microorganism plays oral cavity health care and makees
With.Serious mucosa infection, tumor operation etc. are serious to oral mucosa wound, often cause biggish reconstruction of oral defect, adopt in the past
With iodoform oil yarn packet flap coverage is beaten, neighbouring valve Shift recovery such as buccal fat pad graft, palate valve, neck width muscle flap etc. not only increases operation
Difficulty and wound, and the dysfunction and complication for causing donor site different degrees of.Iodoform oil yarn packet flap coverage is beaten, art is caused
Scar deformity afterwards, causes limitation of mouth opening, tongue limitation of activity etc..
Ideal defect of oral mucosa alternative materials should have the following characteristics that 1) low cost;2) long shelf-life;3) user
Just, surgical procedure is simple, is easy to fixed;4) no antigen;5) there is persistence;6) there is flexibility;7) there is screen to bacterium
Barrier effect;8) good to irregular surface of a wound compliance;It 9) will not hyperplasia.The ideal for meeting all conditions above there is no to replace at present
For object.
People's amnion is located at the innermost layer of placenta, is the semi-transparent film of thickness 20-500um, possesses superior extracellular matrix
Ingredient is acknowledged as having revolutionary improved biomaterial.People takes off cell amnion as a kind of special extracellular matrix, takes
Material is easier to, and quantity is more.It is a kind of de- cell material that people, which takes off cell amnion, and main component is fibronectin splicing variants, layer adhesion egg
White, type Ⅳ collagen, heparan sulfate proteoglycan and growth factor have been widely used in each site tissue damage reparation, such as angle
The fields such as reparation and peripheral nerve regeneration after film transplanting, defect of skin, cartilage damage, and obtain good therapeutic effect.It is real
It tramples and is proved people to take off cell amnion to be a kind of good surface of a wound covering dressing, with good biocompatibility, stability, also
With certain blood vessel elasticity, plastic deformation, anti-inflammatory, anti-infective, nontoxic, low immunogenicity and antigenicity, without oncogenicity etc..
There are many preparation method that people takes off cell amnion, we are comprehensively compared using a kind of quickly and effectively amnion of low cost and takes off cell
Method had both avoided excessively enzymatic hydrolysis destruction in de- cell amnion preparation process compared with traditional pancreatin degradation or physical method
The protein structure of amnion stroma and the possibility of toughness also avoid instrument processing damage caused by amnion.However, de- thin
Born of the same parents' amnion is monolayer amniotic membrane, and Shortcomings are: tissue is thin, mechanicalness is poor, tearable, easy curling, it is difficult to cut forming, cover
It is soluble after the lid surface of a wound.Previous people are increased to overcome above-mentioned deficiency using bilateral/multilayer machinery covering or with collagen binder
Add the thickness of de- cell amnion.Such method is the simple thickness for increasing de- cell amnion, does not have hydrogel
The function of associated advantages such as water imbibition, water-retaining property, gas permeability, flexibility and slow releasing pharmaceutical, to limit its application.
Hydrogel is that the cross-linked network of hydrophilic polymer can expand in water, to obtain many of its original quality
Times.The physics and biochemical property of hydrogel depend primarily on they composition, for they polymerize method and they
Crosslinking degree.In organizational project application aspect, hydrogel can be directed to the design requirement of different biomedical applications, provide one
A multi-functional platform.It has been able to prepare using different cross-linking methods numerous by natural and/or synthetic polymer at present
Hydrogel as regenerative medicine, drug delivery and tissue adhesive.Due to the difference of physicochemical property, common hydrogel is usual
The requirement of intensity and mechanical adjustable is not achieved, and GelMA is with easy acquisition, light is controllable and degradable, has good biology
Compatibility, low immunoreactivity and good mechanical strength, the characteristics such as local sticking nature is good, can be used as bionics prosthesis film and are attached to
Oral mucosa damage surface.But GelMA hydrogel itself lacks bioactive substance, cannot promote the fast of oral mucosa damage
Speed is repaired, and the reparation that GelMA hydrogel is used alone applied to oral mucosa damage is very few, more has no that GelMA passes through electrospinning, light
It is crosslinked the invention report that de- cell amnion prepares Double sided biological active dressing.
Electrostatic spinning technique is a kind of spinning technique for preparing polymer nanofiber developed in recent years, maximum
Feature is that obtained timbering material has many advantages, such as that small porosity height, fibre diameter, large specific surface area, homogeneity are good, heavier
What is wanted is that obtained nanofiber can imitate natural extracellular matrix from micro-scale.It can be made by photo-crosslinking simultaneously
Nanofiber obtained above forms the finer and close crosslinked of three-dimensional net structure, stronger, improves the stabilization of material
Property.GelMA hydrogel/people prepared by the present invention takes off the three-dimensional double-deck dressing of cell amnion, and the biology for remaining de- cell amnion is living
Property, while there is the mechanical strength of GelMA hydrogel.The dressing has the porous space of three-dimensional network shape, similar to natural thin
Extracellular matrix, have bioactivity, can stimulating cellular growth and vascularization, so as to promote the fast of defect of oral mucosa
Speed is repaired.
Summary of the invention
The present invention is directed to disclose a kind of existing good protection effect, has many advantages, such as anti-inflammatory, anti-infective and promote healing, again
Biological active dressing with good gas permeability, water-retaining property and toughness can reach the growth of stimulation defective region cell and blood vessel
It is formed, promotes the quick reparation of defect of oral mucosa, after the regeneration of defective region mucous membrane tissue, former dressing is biodegradable or newborn
Organize the preparation method of the double-deck dressing of the three-dimensional of creeping substitution purpose.
To achieve the goals above, the claimed technical solution of the present invention is as follows:
A kind of GelMA hydrogel people takes off the preparation method of the three-dimensional double-deck auxiliary material of cell amnion, comprising the following steps:
Step 1: the preparation of de- cell amnion (D-HAM): selection hepatitis virus antigen, syphilis antibody and human immunodeficiency virus
Antibody is the puerpera of negative caesarean birth, and fetal membrane is taken under sterile working, and blunt separation removes villus membrane tissue, retains amnion
Amnion is transferred to repeated flushing in the sterile PBS for being supplemented with antibiotic and antibacterial agent and removes blood clot, then with 0.2% by layer
EDTA is handled 30 minutes at 37 DEG C, is then impregnated 30 seconds in 0.5mol/L sodium hydroxide solution, is hereafter transferred into 5%
It is acutely rocked in ammonium chloride, removes cell from amnion by acutely shaking and scraping, finally washed three times with PBS, after freeze-drying
It is spare;
Step 2: 20g gelatin the preparation of GelMA hydrogel electrospinning liquid: is mixed to 60 DEG C of water-baths for being put into preheating with 200mlPBS
Middle stirring and dissolving is dripped 16ml methacrylic anhydride with the speed of 4ml/min with pump to transparent yellow liquid under the conditions of being protected from light
Enter aforesaid liquid and be sufficiently stirred 2 hours, preheated 800mlPBS is added and is stirred to react 15min injection 14-16KD bag filter
It is middle to use deionized water dialysis one week, solution in bag filter will be taken out after a week, be put into -20 DEG C of ice after selecting the filtering of 0.45um filter paper
It case pre-freeze 12 hours, is lyophilized later spare;
Step 3: the preparation of three-dimensional bilayer dressing: take 100mg-120mg that 1ml hexafluoroisopropanol is added to be made into electrospinning liquid, by step 2
De- cell amnion after middle freeze-drying is fixed on progress electrostatic spinning operation, 70% second of the D-HAM/E-GelMA being spun on roller
Alcohol is handled 1 hour, and photoinitiator Irgacure2959 row photo-crosslinking is added in the dressing later, and the dressing after photo-crosslinking is used
PBS cleaning is lyophilized after removing photoinitiator, in γ irradiation sterilization, encapsulates under aseptic condition, and 4 DEG C save up to GelMA water-setting
Glue people takes off the three-dimensional double-deck auxiliary material of cell amnion.
Further improvement of the present invention, the pH value of above-mentioned PBS are 7.4.
Further improvement of the present invention, in above-mentioned steps three, voltage remains 18kV/cm in electrostatic spinning operating process,
The distance between syringe and collector 15cm, constant flow rate 0.3ml/H.
The present invention is suitable for the reparation or temporary mucous membrane substitution of the defect of oral mucosa surface of a wound caused by a variety of causes;By
It is similar with the mechanism that Mucosa Defect heals in defect of skin, it is suitable for skin tissue defects, bedsore or skin
Chronic ulcer.
The present invention can need to configure when necessary containing antimicrobial, hemostatic or analgesic in clinical use according to the surface of a wound
Dressing is impregnated using preceding in above-mentioned medical fluid, is covered in defect of oral mucosa area after its swelling to adsorb drug, so by the medical fluid of medicine
After be fixed, absorbed naturally to it or by hand take out.
Beneficial effects of the present invention: one, GelMA hydrogel passes through Electrospinning and photo-crosslinking and de- cell amnion knot
It closes, forms nanofiber grade three-dimensional network porous structure, it is thin to be conducive to defective region tissue for altitude simulation natural extracellular matrix structure
The growth of born of the same parents.
Two, the bilayer dressing had both had good mechanical strength and compliance, while having the biology of de- cell amnion living
Property, the regenerative healing that defective region can not only be promoted to organize, while stable cell can be provided for cell growth, vascularization
External environment.
If antimicrobial, hemostatic or anodyne, the spatial network energy which has three, are added when dressing uses
It is enough that by drug, slowly the lasting surface of a wound that is discharged into plays the role of continued treatment.
Specific embodiment
In order to deepen the understanding of the present invention, below in conjunction with embodiment, the invention will be described in further detail, the reality
It applies example for explaining only the invention, protection scope of the present invention is not constituted and limited.
Embodiment: a kind of GelMA hydrogel people takes off the preparation method of the three-dimensional double-deck auxiliary material of cell amnion, including following step
It is rapid:
Step 1: the preparation of de- cell amnion (D-HAM): selection hepatitis virus antigen, syphilis antibody and human immunodeficiency virus
Antibody is the puerpera of negative caesarean birth, and fetal membrane is taken under sterile working, and blunt separation removes villus membrane tissue, retains amnion
Amnion is transferred to repeated flushing in the sterile PBS for being supplemented with antibiotic and antibacterial agent and removes blood clot, then with 0.2% by layer
EDTA is handled 30 minutes at 37 DEG C, is then impregnated 30 seconds in 0.5mol/L sodium hydroxide solution, is hereafter transferred into 5%
It is acutely rocked in ammonium chloride, removes cell from amnion by acutely shaking and scraping, finally washed three times with PBS, after freeze-drying
It is spare;
Step 2: 20g gelatin the preparation of GelMA hydrogel electrospinning liquid: is mixed to 60 DEG C of water-baths for being put into preheating with 200mlPBS
Middle stirring and dissolving is dripped 16ml methacrylic anhydride with the speed of 4ml/min with pump to transparent yellow liquid under the conditions of being protected from light
Enter aforesaid liquid and be sufficiently stirred 2 hours, preheated 800mlPBS is added and is stirred to react 15min injection 14-16KD bag filter
It is middle to use deionized water dialysis one week, solution in bag filter will be taken out after a week, be put into -20 DEG C of ice after selecting the filtering of 0.45um filter paper
It case pre-freeze 12 hours, is lyophilized later spare;
Step 3: the preparation of three-dimensional bilayer dressing: take 100mg-120mg that 1ml hexafluoroisopropanol is added to be made into electrospinning liquid, by step 2
De- cell amnion after middle freeze-drying is fixed on progress electrostatic spinning operation, 70% second of the D-HAM/E-GelMA being spun on roller
Alcohol is handled 1 hour, and photoinitiator Irgacure2959 row photo-crosslinking is added in the dressing later, and the dressing after photo-crosslinking is used
PBS cleaning is lyophilized after removing photoinitiator, in γ irradiation sterilization, encapsulates under aseptic condition, and 4 DEG C save up to GelMA water-setting
Glue people takes off the three-dimensional double-deck auxiliary material of cell amnion.
In the present embodiment, the pH value of above-mentioned PBS is 7.4, and voltage remains in electrostatic spinning operating process in step 3
18kV/cm, the distance between syringe and collector 15cm, constant flow rate 0.3ml/H.
Basic principles and main features and advantage of the invention have been shown and described above.The technical staff of the industry should
Understand, the present invention is not limited to the above embodiments, and the above embodiments and description only describe originals of the invention
Reason, without departing from the spirit and scope of the present invention, various changes and improvements may be made to the invention, these changes and improvements
It all fall within the protetion scope of the claimed invention.The claimed scope of the invention is by appended claims and its equivalent circle
It is fixed.
Claims (3)
1. the preparation method that a kind of GelMA hydrogel people takes off the three-dimensional double-deck auxiliary material of cell amnion, which is characterized in that including following step
It is rapid:
Step 1: the preparation of de- cell amnion (D-HAM): selection hepatitis virus antigen, syphilis antibody and human immunodeficiency virus
Antibody is the puerpera of negative caesarean birth, and fetal membrane is taken under sterile working, and blunt separation removes villus membrane tissue, retains amnion
Amnion is transferred to repeated flushing in the sterile PBS for being supplemented with antibiotic and antibacterial agent and removes blood clot, then with 0.2% by layer
EDTA is handled 30 minutes at 37 DEG C, is then impregnated 30 seconds in 0.5mol/L sodium hydroxide solution, is hereafter transferred into 5%
It is acutely rocked in ammonium chloride, removes cell from amnion by acutely shaking and scraping, finally washed three times with PBS, after freeze-drying
It is spare;
Step 2: 20g gelatin the preparation of GelMA hydrogel electrospinning liquid: is mixed to 60 DEG C of water-baths for being put into preheating with 200mlPBS
Middle stirring and dissolving is dripped 16ml methacrylic anhydride with the speed of 4ml/min with pump to transparent yellow liquid under the conditions of being protected from light
Enter aforesaid liquid and be sufficiently stirred 2 hours, preheated 800mlPBS is added and is stirred to react 15min injection 14-16KD bag filter
It is middle to use deionized water dialysis one week, solution in bag filter will be taken out after a week, be put into -20 DEG C of ice after selecting the filtering of 0.45um filter paper
It case pre-freeze 12 hours, is lyophilized later spare;
Step 3: the preparation of three-dimensional bilayer dressing: take 100mg-120mg that 1ml hexafluoroisopropanol is added to be made into electrospinning liquid, by step 2
De- cell amnion after middle freeze-drying is fixed on progress electrostatic spinning operation, 70% second of the D-HAM/E-GelMA being spun on roller
Alcohol is handled 1 hour, and photoinitiator Irgacure2959 row photo-crosslinking is added in the dressing later, and the dressing after photo-crosslinking is used
PBS cleaning is lyophilized after removing photoinitiator, in γ irradiation sterilization, encapsulates under aseptic condition, and 4 DEG C save up to GelMA water-setting
Glue people takes off the three-dimensional double-deck auxiliary material of cell amnion.
2. GelMA hydrogel people according to claim 1 takes off the preparation method of the three-dimensional double-deck auxiliary material of cell amnion, feature
It is, the pH value of the PBS is 7.4.
3. GelMA hydrogel people according to claim 1 or 2 takes off the preparation method of the three-dimensional double-deck auxiliary material of cell amnion,
It is characterized in that, in the step 3, voltage remains 18kV/cm, syringe and collector in the electrostatic spinning operating process
The distance between 15cm, constant flow rate 0.3ml/H.
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Cited By (10)
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CN109876193A (en) * | 2019-04-01 | 2019-06-14 | 西安交通大学 | A kind of two-layer compound paradental defect repair materials and preparation method thereof based on photocrosslinkable hydrogel |
CN110903510A (en) * | 2019-10-08 | 2020-03-24 | 刘明艳 | Preparation method of visible light copolymerization crosslinked porous mesh GelMA-dHAMMA hydrogel |
CN112569399A (en) * | 2020-12-11 | 2021-03-30 | 中山大学 | Photo-crosslinking/electrostatic spinning preparation and application of hydrogel composite scaffold with double-layer skin structure |
CN112675360A (en) * | 2020-12-11 | 2021-04-20 | 中山大学 | Preparation and application of hADSCs-loaded double-layer skin bionic hydrogel composite scaffold |
CN112717208A (en) * | 2020-12-03 | 2021-04-30 | 唐山市工人医院 | Composite multi-layer amnion material for preventing adhesion |
CN113209388A (en) * | 2021-03-30 | 2021-08-06 | 复旦大学附属眼耳鼻喉科医院 | Fibrous membrane for transmitting sound and inducing regeneration of tympanic membrane as well as preparation method and application of fibrous membrane |
CN113456895A (en) * | 2021-07-20 | 2021-10-01 | 广州市朴道联信生物科技有限公司 | GelMA-collagen double-network antibacterial cornea repair material and preparation method and application thereof |
CN114796596A (en) * | 2022-04-22 | 2022-07-29 | 青岛大学 | Nanofiber hydrogel dressing loaded with salvia miltiorrhiza and radix puerariae and preparation process thereof |
CN115845141A (en) * | 2023-03-02 | 2023-03-28 | 健诺维(成都)生物科技有限公司 | Preparation method and application of dry amnion |
CN115869453A (en) * | 2021-09-26 | 2023-03-31 | 中国科学院理化技术研究所 | Double-layer antibacterial dressing loaded with antibacterial molecules, preparation and application |
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Cited By (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109876193A (en) * | 2019-04-01 | 2019-06-14 | 西安交通大学 | A kind of two-layer compound paradental defect repair materials and preparation method thereof based on photocrosslinkable hydrogel |
CN110903510A (en) * | 2019-10-08 | 2020-03-24 | 刘明艳 | Preparation method of visible light copolymerization crosslinked porous mesh GelMA-dHAMMA hydrogel |
CN112717208A (en) * | 2020-12-03 | 2021-04-30 | 唐山市工人医院 | Composite multi-layer amnion material for preventing adhesion |
CN112569399A (en) * | 2020-12-11 | 2021-03-30 | 中山大学 | Photo-crosslinking/electrostatic spinning preparation and application of hydrogel composite scaffold with double-layer skin structure |
CN112675360A (en) * | 2020-12-11 | 2021-04-20 | 中山大学 | Preparation and application of hADSCs-loaded double-layer skin bionic hydrogel composite scaffold |
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