CN107412868A - A kind of preparation method of acellular dermal matrix and obtained acellular dermal matrix - Google Patents
A kind of preparation method of acellular dermal matrix and obtained acellular dermal matrix Download PDFInfo
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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
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
- A61L27/362—Skin, e.g. dermal papillae
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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
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- 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
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
- A61L27/3687—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents
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- 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
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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
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/34—Materials or treatment for tissue regeneration for soft tissue reconstruction
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Abstract
The present invention relates to tissue engineering technique field, more particularly to a kind of preparation method of acellular dermal matrix and obtained acellular dermal matrix.The preparation method of acellular dermal matrix provided by the invention can remove cell component totally, and antigenicity is low, remains dermal collagen structure, allogeneic dna sequence DNA content is extremely low, and has good mechanical property.
Description
Technical field
The present invention relates to tissue engineering technique field, more particularly to a kind of preparation method of acellular dermal matrix and it is made
Acellular dermal matrix.
Background technology
Skin is the vital tissue organ of covering, protection body surface and internal organ, due to inflammation, ulcer wound, burn, Tumor Resection
Afterwards and the reason such as congenital abnormality often results in the defect and exception of skin.Autologous skin graft is used for this tissue defect at present more
Or flap suppresses, but this method not only causes new Traumatic wound, is also usually present donor source limitation.In addition, transplanting
Color and luster, quality, difference functionally be present in the skin that thing often closes on transplantation site.And allosome or xenogenesis skin flap coverage are only
It is temporary, finally stills need autologous transplanting skin.Also, also there is limited source, expensive and have in allosome or xenogenesis skin
Propagate the possibility of pathogenic microorganism and be related to ethics problem etc..
It is current defect of skin using organization engineering skin reparing skin defect with going deep into for tissue engineering technique research
One of focus of repairing research.Acellular dermal matrix (acellular dermal matrix, ADM) is that most have application at present
The dermal substitute of prospect, it eliminates the cell component for triggering host rejection reaction, completely remains extracellular matrix
Morphosis and constituent, mechanical property is good, and antigenicity is low, and it is thin to can induce the fibroblast with power of regeneration, epidermis
Born of the same parents, vascular endothelial cell grow into skin corium according to due histologic patterns, and clinical application effect is preferable.
But existing de- cell pigskin technology still exist it is de- cell is imperfect, has the shortcomings that certain immunogenicity,
Cause clinical practice by a definite limitation, thus clinically there is an urgent need to a kind of low immunogenicity, be available for transplanting skin substitutes
Thing.
The content of the invention
In view of this, the technical problem to be solved in the present invention be the preparation method that a kind of acellular dermal matrix is provided and
Obtained acellular dermal matrix, it is more complete that dermal matrix provided by the invention takes off cell.
The preparation method of acellular dermal matrix provided by the invention, comprises the following steps：
After malicious split-thickness skin graft go out with degreasing agent degreasing, cell is taken off through de-cell liquid, obtains product A；
After the product A is crosslinked with oleuropein Solutions Solution, rinsed successively through alcohol, PBS solution rinses, dries, spoke
According to sterilization, product B is obtained；
After the product B is handled with carboxymethyl chitosan solution, rinsed through water, obtain product C；
After the product C is soaked with glutaraldehyde solution, through softening, moisturizing processing, again with the rinsing of water physiology salt, inactivation of virus
Afterwards, obtained acellular dermal matrix is freezed；
The degreasing agent includes water and following mass fraction：
Soda ash 1.0%~4.0%；
Caustic soda 1.0%~4.0%；
Surfactant 0.1%~1.0%；
The surfactant is selected from peregal, sodium dodecyl aminopropionitrile, AEO or Qula ketone；
The de-cell liquid includes water and following mass fraction：
Pancreatin 0.1%~0.5%；
Activator 0.1%~0.3%；
Degreasing agent 0.2%~0.6%；
The activator is ammonium sulfate；The degreasing agent is peregal or sodium dodecyl sulfate solution.
In the present invention, the time of the degreasing is 30~60min；The quality of the go out malicious split-thickness skin graft and the degreasing agent
Than for 1:4~7.
In some embodiments, the degreasing agent includes water and following mass fraction：
Soda ash 2.0%；
Caustic soda 2.0%；
Peregal 0.5%.
In the embodiment, the mass ratio of go out malicious split-thickness skin graft and the degreasing agent is 1:5.
In some embodiments, the degreasing agent includes water and following mass fraction：
Soda ash 1.0%；
Caustic soda 1.0%；
Sodium dodecyl aminopropionitrile 0.1%.
In the embodiment, the mass ratio of go out malicious split-thickness skin graft and the degreasing agent is 1:4.
In some embodiments, the degreasing agent includes water and following mass fraction：
Soda ash 4.0%；
Caustic soda 4.0%；
Qula ketone 1.0%.
In the embodiment, the mass ratio of go out malicious split-thickness skin graft and the degreasing agent is 1:7.
In the present invention, the cell free time is 30~60min；The malicious split-thickness skin graft that goes out through degreasing takes off with described
The mass ratio of cell liquid is 1:4~7.
In some embodiments, the de-cell liquid includes water and following mass fraction：
In this embodiment, the mass ratio of go out malicious split-thickness skin graft and de-cell liquid through degreasing is 1:5.
In some embodiments, the de-cell liquid includes water and following mass fraction：
Pancreatin 0.1%；
Ammonium sulfate 0.1%；
Lauryl sodium sulfate 0.2%.
In this embodiment, the mass ratio of go out malicious split-thickness skin graft and de-cell liquid through degreasing is 1:4.
In some embodiments, the de-cell liquid includes water and following mass fraction：
Pancreatin 0.5%；
Ammonium sulfate 0.3%；
Peregal 0.5%.
In this embodiment, the mass ratio of go out malicious split-thickness skin graft and de-cell liquid through degreasing is 1:7.
In the embodiment of the present invention, the mass fraction for being crosslinked oleuropein in the oleuropein aqueous solution used is 5%
~10%；The product A and oleuropein solution mass ratio are (1.5~5.0):1；The temperature of the crosslinking is 30~40
℃；Time is 48~72h.
In some embodiments, the mass fraction for being crosslinked oleuropein in the oleuropein solution used is 5%；This reality
Apply in example, the mass ratio of the product A and oleuropein solution is 2:1；The temperature of the crosslinking is 30 DEG C；Time is 48h.
In some embodiments, the mass fraction for being crosslinked oleuropein in the oleuropein solution used is 70%；This
In embodiment, the mass ratio of the product A and oleuropein solution is 3:1；The temperature of the crosslinking is 37 DEG C；Time is 60h.
In some embodiments, the mass fraction for being crosslinked oleuropein in the oleuropein solution used is 10%；This
In embodiment, the mass ratio of the product A and oleuropein solution is 5:1；The temperature of the crosslinking is 40 DEG C；Time is 72h.
Irradiation sterilization after crosslinking uses gamma-rays.
In the embodiment of the present invention, in the carboxymethyl chitosan solution mass fraction of carboxymethyl chitosan be 0.5%~
2.5%；The carboxymethyl chitosan solution and product B mass ratio are (1.5~3.0)：1；The temperature of the processing be 25~
45℃；The processing includes：
PH value 5.5~6.0 reacts 30min；
PH value 4.5~5.5 reacts 60~90min；
PH value 6.5~7.0 reacts 75~80min.
In some embodiments, the mass fraction of carboxymethyl chitosan is 0.5% in the carboxymethyl chitosan solution；It is described
Carboxymethyl chitosan solution and product B mass ratio are 2.0：1；The temperature of the processing is 35 DEG C；The processing includes：
PH value 5.7 reacts 30min；
PH value 5.0 reacts 75min；
PH value 6.8 reacts 80min.
In some embodiments, the mass fraction of carboxymethyl chitosan is 1.5% in the carboxymethyl chitosan solution；It is described
Carboxymethyl chitosan solution and product B mass ratio are 1.5：1；The temperature of the processing is 25 DEG C；The processing includes：
PH value 5.5 reacts 30min；
PH value 4.5 reacts 60min；
PH value 6.5 reacts 75min.
In some embodiments, the mass fraction of carboxymethyl chitosan is 2.5% in the carboxymethyl chitosan solution；It is described
Carboxymethyl chitosan solution and product B mass ratio are 3.0：1；The temperature of the processing is 45 DEG C；The processing includes：
PH value 6.0 reacts 30min；
PH value 5.5 reacts 90min；
PH value 7.0 reacts 80min.
After carboxymethyl chitosan solution processing, net waste liquid is put, then by the material after processing with its mass ratio be 4.0：1
Distilled water is washed 2 times, and 15 minutes every time, water is drained after washing.
In the embodiment of the present invention, the product C soaks by 0.2%~1% glutaraldehyde solution of mass fraction, immersion
Temperature is 35~40 DEG C, and the time is 15~30min.
In some embodiments, the product C soaks by 0.2% glutaraldehyde solution of mass fraction, and the temperature of immersion is
35~40 DEG C, time 25min.
In some embodiments, the product C soaks by 0.6% glutaraldehyde solution of mass fraction, and the temperature of immersion is
35~40 DEG C, time 15min.
In some embodiments, the product C soaks by 1% glutaraldehyde solution of mass fraction, and the temperature of immersion is 35
~40 DEG C, time 30min.
The softening agents of the softening is hexadecyltrimethylammonium chloride；The time of the softening is 30~60min.
The NMF of the moisturizing is glycerine；The temperature of the moisturizing processing is 37 DEG C, and the time is 30~60min.
After being handled by glutaraldehyde, immunogenicity is reduced, and collagenous fibril is crosslinked fusion again and recovers original
Three-dimensional collagen scaffold structure, has modified the space structure of collagen.
In the present invention, the skin source for the malicious split-thickness skin graft that goes out comes from pig.
In the embodiment of the present invention, the preparation method for going out malicious split-thickness skin graft includes：By pigskin be made thickness for 0.1~
2.0mm split-thickness skin graft, then soaked successively through Benza, the malicious split-thickness skin graft that goes out is made in the immersion of inactivation of virus solution；
The concentration of the Benza is 1 ‰.
The time of the Benza immersion is 20~40min.Specially 20min, 30min or 40min.
The inactivation of virus solution includes the Peracetic acid that water and mass fraction are 0.1%~2.0% and mass fraction is
1.0%~5.0% sodium chloride.
In some embodiments, the inactivation of virus solution includes water and mass fraction is 1.0% Peracetic acid and quality
Fraction is 3.0% sodium chloride.
In some embodiments, the inactivation of virus solution includes water and mass fraction is 0.1% Peracetic acid and quality
Fraction is 1.0% sodium chloride.
In some embodiments, the inactivation of virus solution includes water and mass fraction is 2.0% Peracetic acid and quality
Fraction is 5.0% sodium chloride.
The time of the inactivation of virus solution immersion is 30~60min.The matter of the split-thickness skin graft and inactivation of virus solution
Amount is than being 1:4~1:7.Specially 1:4、1:5 or 1:7.
Acellular dermal matrix made from preparation method of the present invention.
Application of the acellular dermal matrix in dressing is prepared made from preparation method of the present invention.
The invention provides a kind of preparation method of acellular dermal matrix, this method can remove cell component dry
Only, antigenicity is low, remains dermal collagen structure, allogeneic dna sequence DNA content is extremely low, and has good mechanical property.
Present invention adds crosslinking agent oleuropein, oleuropein can form stabilization, effective crosslinked products.And with
Traditional chemical cross-linking agent is compared, and it also has, and cytotoxicity (genotoxicity) is low, and good biocompatibility and anti-degradation capability are strong
The advantages of.
Present invention adds carboxymethyl chitosan, carboxymethyl chitosan is the derivative of chitosan, by introducing carboxymethyl
Etc. small chemical group, make it that there is good water solubility in neutral and alkaline environment, while do not influence other intrinsic special
Property.In addition, compared with chitosan, carboxymethyl chitosan has physically better and biological characteristics, for promoting bio-compatible
Property, antibiotic property and wound healing etc. have more preferable effect.
Brief description of the drawings
Fig. 1 shows that the form of product is made in embodiment 1~3；
Fig. 2 shows the Histological Study of split-thickness skin graft；Wherein, Fig. 2-a show the Histological Study of undressed split-thickness skin graft；
Fig. 2-b show that the Histological Study of product is made in embodiment 1；
Fig. 3 shows scanning electron microscopic observation result；Wherein, Fig. 3-a show the scanning electron microscopic observation knot of undressed split-thickness skin graft
Fruit；Fig. 3-b show that the scanning electron microscopic observation result of product is made in embodiment 1；
Fig. 4 lets others have a look at the load-deformation curve of normal skin and the acellular dermal matrix of embodiment 1；
Fig. 5 lets others have a look at the normalization stress relaxation curve of normal skin and the acellular dermal matrix of embodiment 1；
Fig. 6 lets others have a look at the normalization creep curve of normal skin and the acellular dermal matrix of embodiment 1.
Embodiment
The invention provides a kind of preparation method of acellular dermal matrix and obtained acellular dermal matrix, this area
Technical staff can use for reference present disclosure, be suitably modified technological parameter realization.In particular, all similar replacements
Apparent to those skilled in the art with changing, they are considered as being included in the present invention.The method of the present invention
And application is described by preferred embodiment, related personnel can substantially not depart from present invention, spirit and model
Enclose it is interior methods herein and application are modified or suitably changed with combining, to realize and using the technology of the present invention.
Examination material, the instrument that the present invention uses are all common commercially available product, can all be bought in market.
The oleuropein solution that the present invention uses is extracts from plant (jasmine, daphne lilac, lilac, olive)
Extract, extraction is using ethanol water as solvent, and extract solution is after extracting n-butyl alcohol, with macroreticular resin or silica gel column layer
Analysis, obtain oleuropein solution.
With reference to embodiment, the present invention is expanded on further：
Embodiment 1：The preparation method of Xenogenic acellular dermal matrix
A, split-thickness skin graft technique is prepared：It is skin source from BaMa miniature pig skin, to taking dermatotome to be lost hair or feathers, sterilized, with taking skin
Machine animal skin is made the split-thickness skin graft that thickness is 1.0mm, and split-thickness skin graft is made into tomography skin of different shapes with card punch
Piece；30min, degreasing sterilization are soaked with 1 ‰ Benza；
B, virus inactivation technology：By the split-thickness skin graft Peracetic acid of mass fraction 1.0% and the chlorine of mass fraction 3.0%
The inactivation of virus solution for changing sodium composition inactivate 45 minutes, and the mass ratio of skin graft and inactivation of virus solution is 1:5；
C, degreasing process：Using the soda ash of mass fraction 2.0%, the caustic soda of mass fraction 2.0% and the surface of mass fraction 0.5%
The mixed defatting agent of activating agent composition carries out degreasing 30-60 minutes, and the surfactant is peregal, skin graft and mixed defatting
The mass ratio of agent is 1:5；
D, cell technique is taken off：Using the pancreatin of mass fraction 0.3%, the activator of mass fraction 0.2%, mass fraction 0.3%
The de-cell liquid of degreasing agent composition carries out de- cell 45 minutes, and the activator is the ammonium sulfate of mass fraction 0.2%, the degreasing
Agent is the peregal of mass fraction 0.3%, the lauryl sodium sulfate of mass fraction 0.3%, and the mass ratio of skin graft and de-cell liquid is
1:5；
E, oleuropein is crosslinked：The oleuropein solution that use quality score is 5% soaks above-mentioned de- at 30 DEG C
Cell pigskin matrix 48 hours, is then rinsed with alcohol, then is rinsed with PBS, is dried；
F, irradiation sterilization：By above-mentioned de- cell pigskin through gamma-ray irradiation sterilization；
G, compound carboxymethyl chitosan：The PADM made is put into rotary drum, will be 2.0 with PADM quality of materials ratio：1、
35 DEG C of temperature, the carboxymethyl chitosan solution of pH value 5.7, rotary drum is added by several times, reaction adjusts pH value to 5.0 after 30 minutes, then
Reaction 75 minutes, regulation pH value react 80min to 6.8, put net waste liquid after being disposed, then by the material after processing use and its
Mass ratio is 4.0：1 distilled water is washed 2 times, and 15 minutes every time, water is drained after washing；
H, it is the pigskin two-sided immersion handled through carboxymethyl chitosan is molten in the glutaraldehyde that fresh volumetric concentration is 0.2%
25min is crosslinked in liquid, physiological saline fully rinses；Immunogenicity is reduced by the processing, and collagenous fibril is handed over again
Connection fusion recovers original three-dimensional collagen scaffold structure, has modified the space structure of collagen；Pigskin is put into softening agent solution again
Middle immersion 45min carries out softening, is placed into after cleaning in moisturizing agent solution, and 37 DEG C of moisturizings handle 45min, and physiological saline fully rushes
Wash away except residual；
J, lyophilized, packaging and irradiation sterilization technique：Above-mentioned acellular dermal matrix is put into the refrigerating chamber of freeze drier
It is interior it is lyophilized after, shaping packaging, again through gamma-ray irradiation sterilization, as finished product.
Embodiment 2：The preparation method of Xenogenic acellular dermal matrix
A, split-thickness skin graft technique is prepared：It is skin source from BaMa miniature pig skin, to taking dermatotome to be lost hair or feathers, sterilized, with taking skin
Machine animal skin is made the split-thickness skin graft that thickness is 0.1mm, and split-thickness skin graft is made into tomography skin of different shapes with card punch
Piece；20min, degreasing sterilization are soaked with 1 ‰ Benza；
B, virus inactivation technology：By the split-thickness skin graft Peracetic acid of mass fraction 0.1% and the chlorine of mass fraction 1.0%
The inactivation of virus solution for changing sodium composition inactivate 30 minutes, and the mass ratio of skin graft and inactivation of virus solution is 1:4；
C, degreasing process：Using the soda ash of mass fraction 1.0%, the caustic soda of mass fraction 1.0% and the surface of mass fraction 0.1%
The mixed defatting agent of activating agent composition carries out degreasing 30 minutes, and the surfactant is sodium dodecyl aminopropionitrile, skin graft
Mass ratio with mixed defatting agent is 1:4；
D, cell technique is taken off：Using the pancreatin of mass fraction 0.1%, the activator of mass fraction 0.1%, mass fraction 0.2%
The de-cell liquid of degreasing agent composition carries out de- cell 30 minutes, and the activator is the ammonium sulfate of mass fraction 0.1%, the degreasing
Agent is the lauryl sodium sulfate of mass fraction 0.2%, and the mass ratio of skin graft and de-cell liquid is 1:4；
E, oleuropein is crosslinked：The oleuropein solution that use quality fraction is 7.0% soaks above-mentioned de- thin at 37 DEG C
Born of the same parents' pigskin matrix 60 hours, is then rinsed with alcohol, then is rinsed with PBS, is dried；
F, irradiation sterilization：By above-mentioned de- cell pigskin through gamma-ray irradiation sterilization
G, compound carboxymethyl chitosan：The de- cell pigskin made is put into rotary drum, will be with PADM quality of materials ratio
1.5：1st, 25 DEG C of temperature, the carboxymethyl chitosan solution of pH value 5.5, add rotary drum by several times, and reaction adjusts pH value after 30 minutes and arrived
4.5, then react 60 minutes, regulation pH value to 6.5,75min is reacted, put net waste liquid after being disposed, then by the material after processing
It is 4.0 with its mass ratio：1 distilled water is washed 2 times, and 15 minutes every time, water is drained after washing；
H, it is the pigskin two-sided immersion handled through carboxymethyl chitosan is molten in the glutaraldehyde that fresh volumetric concentration is 0.2%
15min is crosslinked in liquid, physiological saline fully rinses；Immunogenicity is reduced by the processing, and collagenous fibril is handed over again
Connection fusion recovers original three-dimensional collagen scaffold structure, has modified the space structure of collagen；Pigskin is put into softening agent solution again
Middle immersion 30min carries out softening, is placed into after cleaning in moisturizing agent solution, and 37 DEG C of moisturizings handle 30min, and physiological saline fully rushes
Wash away except residual；
J, lyophilized, packaging and irradiation sterilization technique：Above-mentioned de- cell pigskin is put into the refrigerating chamber of freeze drier and frozen
After dry, shaping packaging, again through gamma-ray irradiation sterilization, as finished product.
Embodiment 3：The preparation method of Xenogenic acellular dermal matrix
A, split-thickness skin graft technique is prepared：It is skin source from BaMa miniature pig skin, to taking dermatotome to be lost hair or feathers, sterilized, with taking skin
Machine animal skin is made the split-thickness skin graft that thickness is 2.0mm, and split-thickness skin graft is made into tomography skin of different shapes with card punch
Piece；40min, degreasing sterilization are soaked with 1 ‰ Benza；
B, virus inactivation technology：By the split-thickness skin graft Peracetic acid of mass fraction 2.0% and the chlorine of mass fraction 5.0%
The inactivation of virus solution for changing sodium composition inactivate 60 minutes, and the mass ratio of skin graft and inactivation of virus solution is 1:7；
C, degreasing process：Using the soda ash of mass fraction 4.0%, the caustic soda of mass fraction 4.0% and the surface of mass fraction 1.0%
The mixed defatting agent of activating agent composition carries out degreasing 60 minutes, and the surfactant is Qula ketone, skin graft and mixed defatting agent
Mass ratio be 1:7；
D, cell technique is taken off：Using the pancreatin of mass fraction 0.5%, the activator of mass fraction 0.3%, mass fraction 0.5%
The de-cell liquid of degreasing agent composition carries out de- cell 60 minutes, and the activator is the ammonium sulfate of mass fraction 0.3%, the degreasing
Agent is the peregal of mass fraction 0.5%, and the mass ratio of skin graft and de-cell liquid is 1:7；
E, oleuropein is crosslinked：The oleuropein solution that use quality fraction is 10% soaks above-mentioned de- cell at 40 DEG C
Pigskin matrix 72 hours, is then rinsed with alcohol, then is rinsed with PBS, is dried；
F, irradiation sterilization：By above-mentioned de- cell pigskin through gamma-ray irradiation sterilization
G, compound carboxymethyl chitosan：The de- cell pigskin made is put into rotary drum, will be with PADM quality of materials ratio
3.0：1st, temperature 45 C, the carboxymethyl chitosan solution of pH value 6.0, add rotary drum by several times, and reaction adjusts pH value after 30 minutes and arrived
5.5, then react 90 minutes, regulation pH value to 7.0,80min is reacted, put net waste liquid after being disposed, then by the material after processing
It is 4.0 with its mass ratio：1 distilled water is washed 2 times, and 15 minutes every time, water is drained after washing；
H, it is the pigskin two-sided immersion handled through carboxymethyl chitosan is molten in the glutaraldehyde that fresh volumetric concentration is 0.2%
30min is crosslinked in liquid, physiological saline fully rinses；Immunogenicity is reduced by the processing, and collagenous fibril is handed over again
Connection fusion recovers original three-dimensional collagen scaffold structure, has modified the space structure of collagen；Pigskin is put into softening agent solution again
Middle immersion 60min carries out softening, is placed into after cleaning in moisturizing agent solution, and 37 DEG C of moisturizings handle 60min, and physiological saline fully rushes
Wash away except residual；
J, lyophilized, packaging and irradiation sterilization technique：Above-mentioned de- cell pigskin is put into the refrigerating chamber of freeze drier and frozen
After dry, shaping packaging, again through gamma-ray irradiation sterilization, as finished product.
Property Identification
1st, product form such as Fig. 1, the pigskin after processing is milky, and pore is visible, and surface is flat thin smooth.Skin body is soft
Soft, rich in ductility and elasticity, Firmware is strong, is not easy to tear, and can be cut into different shape as requested.
2nd, histological observation：Dyed after product made from embodiment 1~3 is embedded, cut into slices with HE, light microscopic (Japan
Olympus, IX71) tissues observed structural change.As a result as Fig. 2, wherein Fig. 2-a show undressed split-thickness skin graft, it is seen that net
Shape layer dermic hair follice is enriched, rich in cell and blood vessel, arrangement of collagen fibers rule.Fig. 2-b show finished product made from embodiment 1, its
In have no hair in hair follicle, epithelial root sheath residual, the blue dye structure in former capilary region is invisible, do not find to rupture cell residue into
Point, the dicing effect of the finished product of embodiment 2~3 is similar to Fig. 2-b.
3rd, scanning electron microscopic observation：With product made from scanning electron microscopic observation embodiment 1~3, as a result such as Fig. 3, wherein Fig. 3-a
Show undressed split-thickness skin graft, it is seen that the irregular broken sheet-like particle of form, may system do not take off and remain proteoglycan,
The matrix components such as glycoprotein.Fig. 2-b show finished product made from embodiment 1, wherein having no obvious granular material, embodiment 2~3
The dicing effect of finished product is similar to Fig. 2-b.
4th, by after the extracted DNA of the product of embodiment 1~3, DNA content, is tied in fluorescence colorimetric assay detection sample
Fruit table 1.
The measure of table 1DNA contents
Illustrate that skin graft all more can thoroughly remove cell made from embodiment 1~3.
5th, biomechanics experiment：Spindle-type skin test specimen is made in the product of embodiment 1~3, uses universal tensile testing machine
Biomechanics experiment is carried out, setting sensor pressure is 500N, loading velocity 500mm/min, system automatic data collection, is applied
The load-deformation curve, normalization stress relaxation curve, normalization creep that DAS Origin9.0 describes two groups are bent
Line.Abscissa is that Green strains (unit mm/mm) in load-deformation curve, and ordinate is tensile stress (MPa), in stress
Tested in scope (300kPa).Normalize in stress relaxation curve, abscissa is normalization time (s), and ordinate is to return
One changes stress (MPa).Normalize in creep curve, abscissa is normalization time (s), and ordinate is normalization creep (mm/
mm)。
Through biomechanics experiment, it is compacted to depict two groups of load-deformation curve, normalization stress relaxation curve, normalization
Varied curve, as a result show the biomechanical property and normal skin acellular dermal base of ripe cicatricial tissue acellular dermal matrix
Matter is close, sees Fig. 4-6.
It the above is only the preferred embodiment of the present invention, it is noted that come for those skilled in the art
Say, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should be regarded as
Protection scope of the present invention.
Claims (10)
1. a kind of preparation method of acellular dermal matrix, it is characterised in that comprise the following steps：
After malicious split-thickness skin graft go out with degreasing agent degreasing, cell is taken off through de-cell liquid, obtains product A；
After the product A is with oleuropein solution crosslinking, rinsed successively through alcohol, PBS solution rinsing, dry, irradiation sterilization, obtain
Obtain product B；
After the product B is handled with carboxymethyl chitosan solution, rinsed through water, obtain product C；
After the product C is soaked with glutaraldehyde solution, through softening, moisturizing processing, rinsed again with physiological saline, after inactivation of virus,
Lyophilized obtained acellular dermal matrix；
The degreasing agent includes water and following mass fraction：
Soda ash 1.0%~4.0%；
Caustic soda 1.0%~4.0%；
Surfactant 0.1%~1.0%；
The surfactant is selected from peregal, sodium dodecyl aminopropionitrile, AEO or Qula ketone；
The de-cell liquid includes water and following mass fraction：
Pancreatin 0.1%~0.5%；
Activator 0.1%~0.3%；
Degreasing agent 0.2%~0.6%；
The activator is ammonium sulfate；The degreasing agent is peregal or sodium dodecyl sulfate solution.
2. preparation method according to claim 1, it is characterised in that the time of the degreasing is 30~60min；It is described to go out
The mass ratio of malicious split-thickness skin graft and the degreasing agent is 1:4~7.
3. preparation method according to claim 1, it is characterised in that the cell free time is 30~60min；By
The mass ratio of go out malicious split-thickness skin graft and the de-cell liquid of degreasing is 1:4~7.
4. preparation method according to claim 1, it is characterised in that olive in the oleuropein solution that the crosslinking uses
The mass fraction of bitter glycosides is 5%~10%；The product A and oleuropein solution mass ratio are (1.5~5.0)：1；It is described
The temperature of crosslinking is 30~40 DEG C；Time is 48~72h.
5. preparation method according to claim 1, it is characterised in that carboxymethyl chitosan in the carboxymethyl chitosan solution
The mass fraction of sugar is 0.5%~2.5%；The carboxymethyl chitosan solution and product B mass ratio are (1.5~3.0)：1；
The temperature of the processing is 25~45 DEG C；The processing includes：
PH value 5.5~6.0 reacts 30min；
PH value 4.5~5.5 reacts 60~90min；
PH value 6.5~7.0 reacts 75~80min.
6. preparation method according to claim 1, it is characterised in that the product C is using mass fraction as 0.2%~1%
Glutaraldehyde solution immersion, the temperature of immersion is 35~40 DEG C, and the time is 15~30min.
7. according to the preparation method described in any one of claim 1~6, it is characterised in that the skin source of the malicious split-thickness skin graft that goes out
From pig.
8. preparation method according to claim 7, it is characterised in that the preparation method for going out malicious split-thickness skin graft includes：
The split-thickness skin graft that thickness is 0.1~2.0mm is made in pigskin, is then soaked successively through Benza, inactivation of virus solution
The malicious split-thickness skin graft that goes out is made in immersion；
It is 1.0% that the inactivation of virus solution, which includes the Peracetic acid that water and mass fraction are 0.1%~2.0% and mass fraction,
~5.0% sodium chloride.
9. acellular dermal matrix made from any one of claim 1~8 preparation method.
10. application of the acellular dermal matrix in dressing is prepared made from any one of claim 1~8 preparation method.
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Application publication date: 20171201 |