CN105797212A - Preparing method and application of acellular amniotic membrane used for repairing skin wound difficult to heal - Google Patents
Preparing method and application of acellular amniotic membrane used for repairing skin wound difficult to heal Download PDFInfo
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- CN105797212A CN105797212A CN201610255775.2A CN201610255775A CN105797212A CN 105797212 A CN105797212 A CN 105797212A CN 201610255775 A CN201610255775 A CN 201610255775A CN 105797212 A CN105797212 A CN 105797212A
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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
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- A—HUMAN NECESSITIES
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- 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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- 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
- A61L27/3683—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
- A61L27/3691—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 physical conditions of the treatment, e.g. applying a compressive force to the composition, pressure cycles, ultrasonic/sonication or microwave treatment, lyophilisation
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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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
- A61L2430/40—Preparation and treatment of biological tissue for implantation, e.g. decellularisation, cross-linking
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Abstract
The invention relates to a preparing method and application of an amniotic membrane, in particular to a preparing method and application of an acellular amniotic membrane used for repairing skin wound difficult to heal. The preparing method specifically comprises the steps of isolating, cleaning and sterilizing an amniotic membrane tissue; arranging and drying the amniotic membrane tissue on a nitrocellulose membrane to manufacture an amniotic membrane paster; vibrating and digesting the amniotic membrane paster in mixed digestive fluid of 0.25%-0.5% of pancreatin and 0.2-0.5g/L EDTA.4Na for 10-30 minutes at a temperature of 37 DEG C; rinsing the amniotic membrane paster in TE buffer solution; vibrating overnight to fully remove amniotic membrane cells in TE-TritonX-100 solution; washing the amniotic membrane paster for three times in TE solution; vibrating and degrading the amniotic membrane paster for 2-4 hours in nucleic acid degrading solution at a temperature of 37 DEG C; washing the amniotic membrane paster for three times in the TE solution. The prepared acellular amniotic membrane paster can be stored for a long time after being refrigerated, dried, packed and sterilized by cobalt 60 irradiation and is a tissue engineering material which can be taken and used when needed. The acellular amniotic membrane paster can be easily separated from the nitrocellulose membrane to be used for repairing the skin wound after rewatered.
Description
Technical field
The present invention relates to a kind of amniotic membrane preparation method and application.
Background technology
The discarded amnion tissue of perinatal stage is one of important regenerative medicine seed cell source, its wide material sources, is prone to
Obtain and dispute on without ethics, being study hotspot in recent years.Amniotic membrane, as a kind of Immune privilege biograft, has anti-
Scorching, tissue adhesion, eases the pain, and promotes the biological characteristicses such as epithelization, started as far back as 1910 to be just applied to fire victim and
Other surgical operations, are used for promoting cutization, ease the pain and prevent to infect.In recent years, along with the further cognition to amniotic membrane
And amniotic membrane preserves the development of technology of preparing, amniotic membrane becomes research and the focus of application again, is widely used in cornea reconstruction, whole
The fields such as shape, burn treatment.The clinical trial utilizing people's amniotic membrane to carry out repair in trauma is retrieved by America NI H clinical database
There are more than 30 examples, wherein contain the multiple refractory such as diabetic foot ulcer, venous ulcer, skin injury, skin burn, ambustio corneae
More wound.This shows that people's amniotic membrane and characteristics of amniotic extracellular matrix have good application as repair in trauma tissue engineering material
Prospect.
People's amniotic membrane is allogeneic natural degradable material, has good cell and histocompatibility, without vascular stroma
In be conducive to wound containing the composition such as I, type Ⅳ collagen albumen, laminin，LN, fibronectin splicing variants and sulfate-proteoglycan
The sticking of pericyte, divide a word with a hyphen at the end of a line and epithelization, prevent apoptosis, and non-immunogenicity.Live rich in multiple endogenous biological in it
Sex factor, such as basic fibroblast growth factor (bFGF), PDGF (PDGF), VEGF
(VEGF), angiogenesis factor, transforming growth factor β (TGF-β), tissue metal proteases inhibitive factor 1 (TIMP-1), tissue
TIMP 2 (TIMP-2), hepatocyte growth factor (HGF) etc., can promote epithelium formation, hypertrophy, make wound
Mouth quickly-healing；Inflammatory reaction is reduced by removing inflammatory cell；Suppression fibrous tissue hyperplasia, reduces cicatrix.The most permissible
It is used as the covering of wound surface, it is also possible to implant wound, makes the organization engineering skin guiding skin regeneration.
Amniotic membrane is as skin tissue repair material at present, and conventional technology is fresh amnion or deep-frozen amniotic membrane, but
Still suffer from variant cell introduce immunogenicity and preserve inconvenience cannot the feature such as ready access upon use.Therefore this with human acellular amniotic membrane
Natural extracellular matrix skin regeneration material has become as one of important commercialization strategy as tissue engineering product.But mesh
Front existing amniotic membrane method for removing cells still cannot ensure that cell ensures the integrity of amnion stroma on the basis of removing completely, especially
Basement membrane can be caused serious destruction by it, affects seed cell or the adhesion of internal endogenous cell and migration.Patent publication No.
CN1757717A (preparation method of dried active amnion) uses pancreatin long time treatment to combine cell sleaker or swab stick is sloughed
Chrotoplast, but trypsinization can cause the change of collagen membrane structure even to be degraded for a long time, and physics scraping methods can be tight
Heavily destroy the basement membrane composition damage that amniotic epithelial cells adheres to.Patent publication No. CN102327640A (a kind of human acellular amniotic membrane
Preparation method) use SDS to carry out cell cracking, promote the disengaging of amniotic epithelial cells, but SDS low concentration to limit amniotic membrane thin
The removal efficiency of born of the same parents, but high concentration SDS can cause the destruction of amnion stroma.
Summary of the invention
The present invention is taken off by a kind of amniotic membrane using the techniques such as the concussion of pancreatin, detergent, physics, nucleolysis to be combined with each other
Cellular processes, it is thus achieved that can be used for the difficult healedmyocardial skins such as diabetic foot ulcer, venous ulcer, widespread skin defect, skin burn
The reparation of skin damage, the method can ensure amnion stroma structural integrity unlike other human acellular amniotic membrane preparation methoies
On the premise of, reach the purpose that cell completely disengages from, and through biological safety and efficiency evaluation, it is de-thin that the method obtains
Born of the same parents' amniotic membrane avirulence, can degradation in vivo, and can reach to promote difficult healing skin wound surface regeneration and the purpose repaired.
A kind of human acellular amniotic membrane preparation method for skin difficulty healing wound repair of the present invention, it is according to following step
Suddenly carry out:
One, selecting mature Placenta Hominis, blunt separation amnion tissue, normal saline cleans 2～3 times, is subsequently placed at containing antibiotic
Normal saline soaks 10min disinfection；
Two, amnioic epithelium is faced up it is laid on aseptic nitrocellulose membrane, aseptically hang 1～2 hour, system
Become amniotic membrane patch；
Three, amniotic membrane patch is placed in quality concentration expressed in percentage by volume is 0.25%～0.5% trypsin and 0.2～0.5g/L
In EDTA 4Na mixture slaking liquid, 37 DEG C of shaking table concussion digestion 10～30min, shaking speed is 70～90rpm/min；
Four, the TE postdigestive amniotic membrane patch of buffer solution for cleaning step 3 2～3 times are used；
Five, the amniotic membrane patch after step 4 being cleaned is soaked in TE-TritonX-100 solution, is 25 DEG C, turns in temperature
Under conditions of speed is 70～90rpm/min, concussion washing 10～16h；
Six, again the amniotic membrane patch of step 5 is cleaned 5～8 times in TE solution；
Seven, the amniotic membrane patch after step 6 being cleaned is placed in nucleolysis solution, temperature under conditions of temperature is 37 DEG C
Bathe 2～4 hours；
Eight, the amniotic membrane patch after step 7 temperature bath being processed is placed in normal saline cleaning 3～5 times；
Nine, the amniotic membrane patch after step 8 being cleaned through lyophilization, encapsulate and radiate and preserve according to after sterilizing.
The present invention comprises following beneficial effect:
The amniotic membrane method for removing cells that the pancreatin of present invention proposition, detergent, physics concussion combine, can remove sheep efficiently
Theca cell, and ensure the integrity of amnion stroma, zoopery proves that the human acellular amniotic membrane that this method obtains can be effective
The Regeneration and Repair promoting difficult healing skin wound surface.Safety evaluatio result shows, this human acellular amniotic membrane can be satisfied with clinic
The treatment of difficult healing skin damage.
(1) amnion tissue epithelial surface is covered on nitrocellulose membrane upward, can aseptically hang 1-2 hour, can make
Amnion tissue is fully fitted with nitrocellulose membrane, it is to avoid amnion tissue and cellulose nitrate membrance separation in de-cell processes, is prepared as
Amniotic membrane patch keep launch shape, amnion tissue can fully contact with de-cell reagent, raising take off cell efficiency.
(2) amnion tissue uses 0.25%～0.5% trypsinization 10-30min, and short time trypsinization does not results in sheep
The degraded of membrane matrix, can make most of amnion cell depart from from substrate simultaneously, or is in unstable coherent condition.Can be
Follow-up cleaning removes amnion cell completely.
(3) after trypsinization, amnion tissue uses TE-TritonX-100 solution to clean, and can efficiently remove remaining thin
Born of the same parents and other impurity components, ensure that the complete structure of amnion stroma simultaneously.
(4) amnion tissue is after removing cell, and in nucleolysis solution, 37 DEG C are reacted 2-4 hour, can effectively degrade
Nucleic acid compositions, removes the immunogenicity of human acellular amniotic membrane further.
(5) amniotic membrane that the present invention uses takes off cell technology, can remove amnion cell completely, it is thus achieved that have intact basement membrane
Amnion stroma material with marshalling collagen structure.This material is freeze-dried, can preserve for a long time after irradiation sterilization, with take with
With.Amnion tissue thickness does not changes with de-cell processes, and no cytotoxicity can the most well be degraded, and heals difficulty
Wound surface has good repair.
Accompanying drawing explanation
Fig. 1 is fresh amnion group human acellular amniotic membrane HE dyeing (× 200) figure；
Fig. 2 is human acellular amniotic membrane HE dyeing (× 200) figure prepared by SDS group amniotic membrane；
Fig. 3 is that to use quality concentration expressed in percentage by volume be that the human acellular amniotic membrane HE for preparing of 0.25% pancreatin group dyes (× 200)
Figure；
Fig. 4 is human acellular amniotic membrane HE dyeing (× 200) figure prepared by pancreatin detergent group；
Fig. 5 is 0.25% trypsinization 30min for using quality concentration expressed in percentage by volume, the human acellular amniotic membrane HE dyeing of preparation
(× 200) figure；
Fig. 6 is 0.25% trypsinization 60min for using quality concentration expressed in percentage by volume, the human acellular amniotic membrane HE dyeing of preparation
(× 200) figure；
Fig. 7 is 0.25% trypsinization 120min for using quality concentration expressed in percentage by volume, the human acellular amniotic membrane HE dye of preparation
Color (× 200) figure；
Fig. 8 is human acellular amniotic membrane HE dyeing (× 400) figure of nucleolysis；
Fig. 9 is human acellular amniotic membrane HE dyeing (× 400) figure of non-nucleolysis；
Figure 10 is that human acellular amniotic membrane treats rat difficulty healing skin wound surface figure.
Detailed description of the invention
Detailed description of the invention one: prepared by a kind of human acellular amniotic membrane for skin difficulty healing wound repair of present embodiment
Method, it is characterised in that it follows the steps below:
One, selecting mature Placenta Hominis, blunt separation amnion tissue, normal saline cleans 2～3 times, removes clot, colloid etc. miscellaneous
Matter composition, is subsequently placed at containing soaking 10min disinfection in antibiotic normal saline；
Two, amnioic epithelium is faced up it is laid on aseptic nitrocellulose membrane, aseptically hang 1～2 hour, make
Amniotic membrane fits tightly with nitrocellulose membrane, makes amniotic membrane patch, and is cut to appropriately sized；
Three, amniotic membrane patch is placed in quality concentration expressed in percentage by volume is 0.25%～0.5% trypsin and 0.2～0.5g/L
In EDTA 4Na mixture slaking liquid, 37 DEG C of shaking table concussion digestion 10～30min, shaking speed is 70～90rpm/min, makes sheep
Theca cell efficiently separates in amnion stroma；
Four, use the TE postdigestive amniotic membrane patch of buffer solution for cleaning step 3 2～3 times, remove and digest in amniotic membrane patch
The thickness colloid separated and cell mass；
Five, the amniotic membrane patch after step 4 being cleaned is soaked in TE-TritonX-100 solution, is 25 DEG C, turns in temperature
Under conditions of speed is 70～90rpm/min, concussion washing 10～16h, fully remove the residual cells in amniotic membrane patch and impurity；
Six, again the amniotic membrane patch of step 5 is cleaned 5～8 times in TE solution, fully remove and reside in going between tissue
Dirty agent composition；
Seven, the amniotic membrane patch after step 6 being cleaned is placed in nucleolysis solution, temperature under conditions of temperature is 37 DEG C
Bathe 2～4 hours, the accounting component of degraded residual, reduce the immunogenicity of amnion stroma further；
Eight, the amniotic membrane patch after step 7 temperature bath being processed is placed in normal saline cleaning 3～5 times, makes amnion tissue complete
Complete clean, without other residues；
Nine, the amniotic membrane patch after step 8 being cleaned through lyophilization, encapsulate and radiate and preserve according to after sterilizing.
Co 60 is used to carry out radiation according to sterilizing.
Detailed description of the invention two: present embodiment is unlike detailed description of the invention one: containing described in step one is anti-
Antibiotic content in raw element normal saline is: concentration be the penicillin of 50mg/L, concentration be streptomycin and the concentration of 50mg/L
Amphotericin B for 2.5mg/L.Other is identical with detailed description of the invention one.
Detailed description of the invention three: present embodiment is unlike detailed description of the invention one: the open assembly time of step 2 is 1
～1.5 hours.Other is identical with detailed description of the invention one.
Detailed description of the invention four: present embodiment is unlike detailed description of the invention one: the open assembly time of step 2 is
1.5～2 hours.Other is identical with detailed description of the invention one.
Detailed description of the invention five: present embodiment is unlike detailed description of the invention one: the open assembly time of step 2 is
1.2～1.6 hours.Other is identical with detailed description of the invention one.
Detailed description of the invention six: present embodiment is unlike detailed description of the invention one: the open assembly time of step 2 is
1.4～1.8 hours.Other is identical with detailed description of the invention one.
Detailed description of the invention seven: present embodiment is unlike detailed description of the invention one: the digestion time of step 3 is
10～20min.Other is identical with detailed description of the invention one.
Detailed description of the invention eight: present embodiment is unlike detailed description of the invention one: the digestion time of step 3 is
15～20min.Other is identical with detailed description of the invention one.
Detailed description of the invention nine: present embodiment is unlike detailed description of the invention one: the digestion time of step 3 is
20～30min.Other is identical with detailed description of the invention one.
Detailed description of the invention ten: present embodiment is unlike detailed description of the invention one: the digestion time of step 3 is
18～25min.Other is identical with detailed description of the invention one.
Detailed description of the invention 11: answering of a kind of human acellular amniotic membrane for skin difficulty healing wound repair of present embodiment
With, it is for preparing the tissue engineering material that skin wound is repaired.
Detailed description of the invention 12: present embodiment is unlike detailed description of the invention 11: described organizational project
During materials'use, carrying out rehydration in sterilized water or normal saline, after rehydration, human acellular amniotic membrane tweezers are from nitrocellulose membrane
Separate, fill and cover to skin injury part, repairing skin wound surfaces.Other is identical with detailed description of the invention 11.
Present invention is not limited only to the content of the respective embodiments described above, one of them or the group of several detailed description of the invention
Contract sample can also realize the purpose of invention.
By following example checking beneficial effects of the present invention:
The agent prescription of following example is as follows:
Pancreatin mixture slaking liquid: quality concentration expressed in percentage by volume is 0.25%～0.5% trypsin；Quality volume basis is dense
Degree is 0.2～0.5g/L EDTA 4Na.
TE buffer: concentration is the TRIS-HCl of 10mM, pH=7.6；Concentration is the EDTA of 1mM.
TE-TritonX-100:TE buffer (pH=7.6)；Concentration expressed in percentage by volume is 0.5%～1%TritonX-100
(v/v)。
Nucleolysis liquid: concentration is the DNase of 30U/mL；Concentration is the Rnase of 10U/mL；Concentration is 50mM/L's
Tris-HCl；Concentration is the MgCl of 10mM/L2；Concentration is the BSA of 30mg/mL；PH=7.6.
Embodiment 1
The present embodiment passes through com-parison and analysis: fresh amnion, SDS process amniotic membrane, 0.25% trypsinization amniotic membrane and this
Bright pancreatin detergent mode processes amniotic membrane, and the amniotic membrane after processing is compared analysis respectively, and amniotic membrane takes off cell state, specifically
As follows:
1) preparation of amniotic membrane patch
Amniotic membrane and nitrocellulose membrane carry out following packet after fitting: unseasoned group, be dried 15 minutes groups, be dried 1 hour group,
It is dried 2 hours groups, is dried 3 hours groups.Amniotic membrane patch carries out above de-cell subsequently process, processing procedure finds, the most dry
Dry group is connected with nitrocellulose membrane loose due to amniotic membrane, is mutually disengaged completely during trypsinization, it is impossible to carry out follow-up de-
Cell technique.It is dried 15 minutes group amniotic membranes and is connected relative loose with nitrocellulose membrane, during trypsinization, have part the most de-
From, it is kept completely separate after detergent-treatment.It is dried 1 hour and 2 hours group amniotic membrane patch and still keeps non-completely dried state, operation
Convenient, in de-cell processes, amniotic membrane is the most closely coupled with nitrocellulose membrane, reaches preferably to take off cell effect.It is dried 3 hours
Group is long due to drying time, and amniotic membrane patch quality becomes fragile, and easily causes tear damage during cutting, brings to de-cell technique
Inconvenience.
2) fresh amnion: after being peeled off on Placenta Hominis by amnion tissue, normal saline cleans 2-3 time, removes blood clot, colloid
Deng impurity, being after 4% paraformaldehyde is fixed by quality concentration expressed in percentage by volume, routine paraffin wax embeds, and HE dyes.
Result is as it is shown in figure 1, result shows that amniotic epithelial cells proper alignment is distributed in basement membrane, mescenchymal stem cell
In the collagen structure of fibroblast layer.Nucleus is that pink can substantially be distinguished for blue, extracellular matrix, and basement membrane is complete
Whole, collagen structure is neat, fine and close.
3) SDS group amniotic membrane: after being cleaned in normal saline by amniotic membrane, epithelial surface is laid on nitrocellulose membrane upward, system
Become amniotic membrane patch.Amniotic membrane patch is placed in 25 DEG C of concussions in TE-0.03%SDS (w/v) mixed liquor and rinses 16 hours, and TE buffer is clear
After washing, it is placed in nucleolysis solution 37 DEG C and reacts 3 hours.After normal saline cleans, by amnion tissue on nitrocellulose membrane
Separating, be that after 4% paraformaldehyde is fixed, routine paraffin wax embeds with weight/mass percentage composition, HE dyes.
Result is as in figure 2 it is shown, result shows, amnionic basement membrane and fibroblast layer all have cell residue
4) 0.25% pancreatin group amniotic membrane: after being cleaned in normal saline by amniotic membrane, epithelial surface is laid in cellulose nitrate upward
On film, make amniotic membrane patch.Amniotic membrane patch is placed in 0.25% trypsin and EDTA 4Na mixture slaking that concentration is 0.2g/L
In liquid, 37 DEG C of concussions digest 2 hours.After TE buffer solution for cleaning, it is placed in nucleolysis solution 37 DEG C and reacts 3 hours.Physiology salt
After water cleans, amnion tissue is separated on nitrocellulose membrane, be after 4% paraformaldehyde is fixed with weight/mass percentage composition, conventional
Paraffin embedding, HE dyes.
Result as it is shown on figure 3, result shows, noresidue cell in amnion tissue, but amnionic basement membrane and fibroblast
Layer all has substantially damage, has cavity generation in collagen structure.
5) pancreatin detergent group (i.e. the method for the present invention): after being cleaned in normal saline by amniotic membrane, epithelial surface is put down upward
It is laid on nitrocellulose membrane, makes amniotic membrane patch.Amniotic membrane patch is placed in TE-0.5%tritonX-100 (v/v) mixed liquor 25
DEG C concussion rinsing 16 hours.After TE buffer solution for cleaning, it is placed in nucleolysis solution 37 DEG C and reacts 3 hours.Normal saline cleans
After, amnion tissue is separated on nitrocellulose membrane, is after 4% paraformaldehyde is fixed by quality concentration expressed in percentage by volume, conventional stone
Wax embeds, and HE dyes.
As shown in Figure 4, result shows result, noresidue cell in amnion tissue, basement membrane and fibroblast Rotating fields
Completely, non-collagen components arrangement is closely, in order.
Embodiment 2
The present embodiment is optimized analysis to amniotic membrane digestion time, specific as follows:
Amniotic membrane patch is placed in quality concentration expressed in percentage by volume and is 0.25% trypsin and concentration is 0.2g/L EDTA 4Na
In mixture slaking liquid, 37 DEG C of concussion digestion 30 minutes, 1 hour, 2 hours respectively, TE buffer solution for cleaning is placed on TE-0.5%
In tritonX-100 (v/v) mixed liquor, 25 DEG C of concussions rinse 16 hours.After TE buffer solution for cleaning, it is placed in nucleolysis solution
37 DEG C are reacted 3 hours.After normal saline cleans, amnion tissue is separated on nitrocellulose membrane, after 4% paraformaldehyde is fixing,
Routine paraffin wax embeds, and HE dyes.
Result as illustrated in figs. 5-7, shows that trypsinization all can remove cell, wherein action time more than 30 minutes completely
The digestion time of 30 minutes ensure that human acellular amniotic membrane has optimal complete structure, as time went on amnionic basement membrane
Damage, non-collagen components is gradually lost.
Embodiment 3
The present embodiment carries out nucleolysis and does not carries out nucleolysis comparative analysis amniotic membrane patch, specific as follows:
Amniotic membrane patch is placed in 0.25% trypsin and 0.2g/L EDTA 4Na mixture slaking liquid, and 37 DEG C of concussions are respectively
Digesting 30 minutes, it is little that TE buffer solution for cleaning is placed on 25 DEG C of concussion rinsings 16 in TE-0.5%tritonX-100 (v/v) mixed liquor
Time.Amniotic membrane patch selects directly to draw materials after cleaning or in nucleolysis solution, 37 DEG C of reactions were drawn materials, by de-cell after 3 hours
After amniotic membrane separates on nitrocellulose membrane, carry out conventional fixing, embedding and HE dyeing.
As Figure 8-9, result shows that human acellular amniotic membrane has bright without the human acellular amniotic membrane surface of nucleolysis to result
Aobvious residual nucleic acid component；Human acellular amniotic membrane can remove nucleic acid component after carrying out nucleolysis completely, removes amniotic membrane further
The immunogenicity of internal transplanting.
Embodiment 4
Thickness sensitivity, concrete operations are as follows:
6) fresh amnion: after being peeled off on Placenta Hominis by amnion tissue, normal saline cleans 2-3 time, removes blood clot, colloid
Deng impurity, blot amniotic membrane surface moisture with filter paper, clamp amniotic membrane with thickness gauge (precision 0.01mm), measure 5-7 diverse location
Amniotic membrane thickness, fresh amnion thickness is 17.8.
7) pancreatin detergent group: after amniotic membrane is cleaned in normal saline, epithelial surface is laid on nitrocellulose membrane upward,
Make amniotic membrane patch.Amniotic membrane patch is placed in 25 DEG C of concussions in TE-0.5%tritonX-100 (v/v) mixed liquor and rinses 16 hours.
After TE buffer solution for cleaning, it is placed in nucleolysis solution 37 DEG C and reacts 3 hours.After normal saline cleans, by amnion tissue from nitre
Separate on acid fibrous membrane, blot amniotic membrane surface moisture with filter paper, clamp amniotic membrane with thickness gauge (precision 0.01mm), measure 5-7
The amniotic membrane thickness of diverse location.
Result is as shown in table 1, and result shows, uses the human acellular amniotic membrane that the present embodiment method obtains compared with fresh amnion
No significant difference, structure does not occurs substantially to change.
Table 1 human acellular amniotic membrane thickness measurement
| Fresh amnion | Human acellular amniotic membrane | |
| Thickness (μm) | 17.8±4.9 | 18.25±3.1 |
。
Embodiment 5
Cytotoxicity detects, and concrete operations are as follows:
8), after amniotic membrane cleans in normal saline, epithelial surface is laid on nitrocellulose membrane upward, makes amniotic membrane patch.Sheep
Film paster is placed in 25 DEG C of concussions in TE-0.5%tritonX-100 (v/v) mixed liquor and rinses 16 hours.After TE buffer solution for cleaning,
It is placed in nucleolysis solution 37 DEG C to react 3 hours.After normal saline cleans, by freeze-dried for human acellular amniotic membrane paster, bag
Dress and 60Coradiation sterilizing.Amniotic membrane patch is separated after rehydration in normal saline on nitrocellulose membrane, takes 40cm2Left and right sheep
In the DF12 culture medium of membrane tissue 5 times of volumes of addition (being the FBS of 10% containing concentration expressed in percentage by volume), 37 DEG C extract 24 hours.Leaching
Extract DF12 culture medium (being the FBS of 10% containing concentration expressed in percentage by volume) dilutes, obtain respectively original content 12.5%, 50%,
The amnion tissue lixiviating solution of 100%, conventional DF12 culture medium (being the FBS of 10% containing concentration expressed in percentage by volume) is as a control group.People
Fibroblast is cultivated respectively in amniotic membrane lixiviating solution and DF12 culture medium, digested at 24 hours, 48 hours, 72 hours respectively and obtains
Obtain cell, use trypan exclusion stain to carry out cell counting.
Cell relative growth rate (RGR)=experimental group cell quantity/cellular control unit quantity × 100%
Result shows, human acellular amniotic membrane prepared by the present embodiment is hypotoxicity or avirulence, meets country the 3rd class medical treatment
Apparatus biological assessment standard (GB/T16886.5-2003).
Table 2 human acellular amniotic membrane in vitro toxicity is evaluated
Embodiment 6
Human acellular amniotic membrane repairs rat difficulty healing skin wound surface, and detailed process is as follows:
9), after amniotic membrane cleans in normal saline, epithelial surface is laid on nitrocellulose membrane upward, makes amniotic membrane patch.Sheep
Film paster is placed in 25 DEG C of concussions in TE-0.5%tritonX-100 (v/v) mixed liquor and rinses 16 hours.After TE buffer solution for cleaning,
It is placed in nucleolysis solution 37 DEG C to react 3 hours.After normal saline cleans, by freeze-dried for human acellular amniotic membrane paster, bag
Dress and 60Coradiation sterilizing.Amniotic membrane patch is cut into a diameter of 2cm2Left and right is circular standby.
10), after the depilation of Wistar rat back, diameter 1.5cm is respectively cut at spinal column two side position2The circular holostrome skin in left and right
Skin, sews up with medical silica-gel circle outside skin wound, manufactures difficult healing skin wound surface.
11) matched group: iodophor disinfection processes, hospital gauze is wrapped up, and a medicine is changed in sterilization in every three days.
12) experimental group: iodophor disinfection processes, by human acellular amniotic membrane and nitric acid after rehydration in amniotic membrane patch physiological saline solution
Fibrous membrane separates, and human acellular amniotic membrane is laid in wound surface, makes amniotic membrane completely attach to wound surface, and hospital gauze is wrapped up, every three days
Sterilization is changed dressings once.
Result as shown in Figure 10, has good repair to difficulty healing wound surface.
Claims (10)
1. the human acellular amniotic membrane preparation method for skin difficulty healing wound repair, it is characterised in that it is according to following step
Suddenly carry out:
One, selecting mature Placenta Hominis, blunt separation amnion tissue, normal saline cleans 2～3 times, is subsequently placed at containing antibiotic physiology
Saline soaks 10min disinfection；
Two, amnioic epithelium is faced up it is laid on aseptic nitrocellulose membrane, aseptically hang 1～2 hour, make sheep
Film paster；
Three, amniotic membrane patch is placed in quality concentration expressed in percentage by volume is 0.25%～0.5% trypsin and 0.2～0.5g/L
In EDTA 4Na mixture slaking liquid, 37 DEG C of shaking table concussion digestion 10～30min, shaking speed is 70～90rpm/min；
Four, the TE postdigestive amniotic membrane patch of buffer solution for cleaning step 3 2～3 times are used；
Five, the amniotic membrane patch after step 4 being cleaned is soaked in TE-TritonX-100 solution, temperature be 25 DEG C, rotating speed be
Under conditions of 70～90rpm/min, concussion washing 10～16h；
Six, again the amniotic membrane patch of step 5 is cleaned 5～8 times in TE solution；
Seven, by step 6 clean after amniotic membrane patch be placed in nucleolysis solution, under conditions of temperature is 37 DEG C temperature bath 2～
4 hours；
Eight, the amniotic membrane patch after step 7 temperature bath being processed is placed in normal saline cleaning 3～5 times；
Nine, the amniotic membrane patch after step 8 being cleaned through lyophilization, encapsulate and radiate and preserve according to after sterilizing.
A kind of human acellular amniotic membrane preparation method for skin difficulty healing wound repair the most according to claim 1, it is special
Levy be described in step one containing the antibiotic content in antibiotic normal saline be: concentration is the penicillin of 50mg/L, dense
Streptomycin and concentration that degree is 50mg/L are the amphotericin B of 2.5mg/L.
A kind of human acellular amniotic membrane preparation method for skin difficulty healing wound repair the most according to claim 1, it is special
Levy and be that the open assembly time of step 2 is 1～1.5 hour.
A kind of human acellular amniotic membrane preparation method for skin difficulty healing wound repair the most according to claim 1, it is special
Levy and be that the open assembly time of step 2 is 1.5～2 hours.
A kind of human acellular amniotic membrane preparation method for skin difficulty healing wound repair the most according to claim 1, it is special
Levy and be that the open assembly time of step 2 is 1.2～1.6 hours.
A kind of human acellular amniotic membrane preparation method for skin difficulty healing wound repair the most according to claim 1, it is special
Levy and be that the open assembly time of step 2 is 1.4～1.8 hours.
A kind of human acellular amniotic membrane preparation method for skin difficulty healing wound repair the most according to claim 1, it is special
Levy and be that the digestion time of step 3 is 10～20min.
A kind of human acellular amniotic membrane preparation method for skin difficulty healing wound repair the most according to claim 1, it is special
Levy and be that the digestion time of step 3 is 15～20min.
9. use an application for the human acellular amniotic membrane for skin difficulty healing wound repair prepared by claim 1, its feature
It is that it is for preparing the tissue engineering material that skin wound is repaired.
The application of a kind of human acellular amniotic membrane for skin difficulty healing wound repair the most according to claim 9, its feature
Be it for burning, Venous Ulcers, diabetic foot or deep dermis damage wound surface reparation.
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