CN108478869A - A kind of Regeneration and Repair bionical matrix of de- cell black carp skin and its preparation method and application - Google Patents
A kind of Regeneration and Repair bionical matrix of de- cell black carp skin and its preparation method and application Download PDFInfo
- Publication number
- CN108478869A CN108478869A CN201810344297.1A CN201810344297A CN108478869A CN 108478869 A CN108478869 A CN 108478869A CN 201810344297 A CN201810344297 A CN 201810344297A CN 108478869 A CN108478869 A CN 108478869A
- Authority
- CN
- China
- Prior art keywords
- cell
- black carp
- carp skin
- regeneration
- repair
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—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
-
- 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
- A61L27/3687—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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/60—Materials for use in artificial skin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- 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
- A61L2430/10—Materials or treatment for tissue regeneration for reconstruction of tendons or ligaments
-
- 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
Abstract
The invention discloses a kind of Regeneration and Repair bionical matrix of de- cell black carp skin and its preparation method and application.The Regeneration and Repair with the bionical matrix of de- cell black carp skin is formed using black carp skin as raw material is specially treated.The Regeneration and Repair, which with the bionical Matrix formulation procedure of de- cell black carp skin includes raw material pre-treatment, thick arrangements, degreasing, de- cell, arrange again, preserves liquid the processing steps such as pre-processes, is molded.Regeneration and Repair prepared by the method for the present invention is thorough with the bionical stroma cell removing of de- cell black carp skin, natural porous network structure is complete, is the bionic extracellular matrix timbering material of low immunogenicity, can provide good microenvironment for regeneration and restoration and cell growth；Compared to the acellular matrix product in the sources such as common pig, ox, the bionical matrix zoonotic virus of de- cell black carp skin prepared by the present invention carries and propagation risk is low, religious ethics risk is low, resourceful and of low cost.
Description
Technical field
The invention belongs to biomedical materials fields, and in particular to a kind of Regeneration and Repair bionical matrix of de- cell black carp skin
And its preparation method and application.
Background technology
The regeneration of wound is that the difficult medical problem solved is badly in need of in many clinical departments always, although perfect with theory of medicine
With the progress of technology, many novel traumas repair product and continue to bring out and applied, but still hardly possible meets growing clinic
Demand.So far, wound repair problem is still to lead to disabled or even dead one of principal element.
Current clinically used wound repair product mainly includes source instrument type, wet union type and bion,
In, bion wound repair product because its have close to organism physiology structure or function biological activity, meet biochemistry group again
It knits reparation to require, has been increasingly becoming most potential wound repair/surface of a wound care product.So far, acellular matrix
(Acellular dermal matrix, ADM) is that the bion wound of closest body tissue physiological structure and function is repaiied
Reproduce product.De- cell pigskin, ox-hide have been developed that into the reparation that medical article is successfully applied to clinical refractory wounds, promote
The function of regeneration and wound healing has also obtained clinical confirmation, but since there are rabid ox diseases (ox), blue otopathy (pig) et al.
Poultry suffers from the risk of viral transmission altogether, has been cited as the product that national Yao Jian general bureaus are included in highest risk class and gives monitor closely.
Further, since the problems such as religious belief, pig, ox source property medical product Israel and other Islam, area not
Can enter clinical application, cause these regions due to the surface of a wound nursing it is improper caused by amputation, infection and the death rate it is high.
Fish-skin is handled in processing of aquatic products as waste resource, and annual discarded amount causes huge ring up to as many as tens thousand of tons
Protect pressure in border.The structure of fish-skin is similar with mammal skin, but the thermal denaturation temperature of collagen, heat-shrinkable compared with
Low, hydroxyproline content is about 10%, and intermolecular cross-linking degree is relatively low, therefore has unique biological activity.In addition, the disease of fish
Poison propagates risk and is far below terricole, can also effectively evade religious ethics barrier, and therefore, it is ideal to take off cell fish-skin matrix
Biomimic materials.But as emerging technology and research direction, the product and technology in the field are few, and only U.S. FDA is criticized so far
Accurate the first de- cell fish-skin product K erecisTMFor the treatment of chronic ulcer, which is de- cell salmon skin,
Containing unsaturated fatty acids such as abundant Ω -3, excellent anti-inflammatory performance is shown, the healing of refractory wounds can be effectively facilitated.And
China is in the research in the field and technology almost blank.
KerecisTMSalmon used belongs to cold water marine fish, and fat content is high, without spy in preparation process
Different ungrease treatment delays routine instead although the equal unsaturated fatty acids of richness Ω -3 can promote the healing of refractory wounds
The healing of the surface of a wound.The fish-skin component and function difference of different fish are very big, biology performance and processing method also big phase diameter
Front yard.China's salmon depends on import, material source not to enrich.Black carp is one of China four freshwater fish, and annual processing generates
Discarded fish-skin resource it is considerable, black carp skin fat content is few, compact structure, and mechanical property is good, and the Oroqen even will be green
Fish-skin is processed into the clothes of resisting cold.Present invention combination China's aquatic products processing situation selects black carp skin as raw material, provides one kind
Preparation method for taking off cell black carp skin Biomimic materials, it is control pyrogen, thin in introducing of the technical process without organic reagent
The pollutions such as bacterium are prepared for a kind of Biomimic materials can be used for a variety of soft tissue regeneration reparations.
Invention content
The present invention provides a kind of Regeneration and Repair bionical matrix of de- cell black carp skin and its preparation method and application, mesh
Be, avoid terricole source property biomaterial viral transmission risk, reduce religious ethics risk, provide it is a kind of have it is good
The de- bionical matrix of cell black carp skin of biological safety, compatibility and mechanical property, the regeneration for being particularly suitable for soft group of wound are repaiied
It is multiple.
To achieve the above object, the present invention uses following technical scheme：
The first aspect of the invention is to provide a kind of Regeneration and Repair preparation method for taking off the bionical matrix of cell black carp skin,
Its main technical schemes is that the Regeneration and Repair is made after the processing such as degreasing, de- cell with de- cell using black carp skin as raw material
The bionical matrix of black carp skin.
Further, the Regeneration and Repair preparation method for taking off the bionical matrix of cell black carp skin, includes the following steps：
(1) black carp skin pre-processes：By the cleaning of fresh black carp skin, removal redundancy tissue and impurity；
(2) mucous membrane/liquid and degreasing are removed：At 4 DEG C, step (1) treated black carp skin carries out thick arranging that remove part superfluous
It is remaining；
(3) micro-structure is protected：At 4 DEG C, with solid-liquid ratio 1:5-1:20 (w/w) phosphate buffer (0.05-0.2M,
PH7.0-7.4) stirring 2-4h heel row removes waste liquid；Above step repeats 1-2 times.
(4) it cleans：At 4 DEG C, with solid-liquid ratio 1:5-1:The 2%-5%NaCl solution stirring 4h heel row of 20 (w/w) removes waste liquid.
(5) de- cell processing for the first time：At 4 DEG C, with solid-liquid ratio 1:5-1:The de- cell solution stir process 12- of 20 (w/w)
20h arranges black carp skin and removes the partial redundance tissue that above-mentioned processing procedure is formed again；At 4 DEG C, with solid-liquid ratio 1:5-1:
The physiological buffer of 20 (w/w) cleans 2-4 times, each 0.5-4h.
(6) micro-structure is protected again：At 4 DEG C, with solid-liquid ratio 1:5-1:20 (w/w) phosphate buffer (0.05-0.2M,
PH7.0-7.4) stirring 2-4h heel row removes waste liquid；Above step repeats 1-2 times.
(7) secondary de- cell processing：Selection handles different de- cell systems and corresponding reaction from first de- cell
Condition carries out secondary de- cell processing, solid-liquid ratio 1:5-1:20(w/w).
(8) it cleans：At 4 DEG C, with solid-liquid ratio 1:5-1:The 2%-5%NaCl solution stirring 4h heel row of 20 (w/w) removes waste liquid.
(9) pretreatment of protection liquid and machine-shaping：At 4 DEG C, the de- bionical matrix of cell black carp skin of preparation is soaked in material
Liquor ratio 1:5-1:In the protection liquid of 20 (w/w), it is slowly stirred 2-8h；According to clinical demand, hygrometric state or dry may be selected in formed product
State：Hygrometric state is to sterilize after the de- bionical matrix of cell black carp skin of infiltration protection liquid is directly packed, sealed；Dry state then protects infiltration
The de- bionical matrix of cell black carp skin of shield liquid is first freeze-dried, then is packed, seals, sterilized.
(10) final sterilization obtains finished product：It is final that the dry state of gained Packing Sound or hygrometric state are taken off into the bionical matrix of cell black carp skin
Sterilizing, can obtain finished product.
It is further preferred that the black carp skin is the material of fresh stripping, which there is trackability in source and meets quarantine, wants
It asks.
It is further preferred that each preparation process temperature control is in 4 DEG C or so (except particular/special requirements), feed liquid in step (1)-(10)
Than 1:5-1:20.
It is further preferred that the pretreated specific treatment process of black carp skin is：By fresh black carp skin scraping off residual squama
The tissues such as piece and redundant muscular, 4 DEG C of deionized waters, which are repeatedly rinsed, eliminates residual blood；And conjunction is cut into according to different anatomic position
Suitable size, is immediately placed in -40 DEG C of freezings, and using preceding taking-up, flowing water thaws at room temperature.
It is further preferred that the removal mucous membrane/liquid and the concrete technology of degreasing are：At 4 DEG C, with solid-liquid ratio 1:5-1:20
(w/w) 2%-5%NaCl solution stirring 1-4h heel row removes waste liquid, and carrying out thick arrange to black carp skin removes partial redundance, above
Step repeats 2-4 times.
It is further preferred that it is 0.5-4% NaTDCs, 0.1-1% dodecanes to take off cell solution described in step (5)
One kind of base sodium sulphate, 0.1-1%Triton X-100, alkyl glycosides.
It is further preferred that it is 0.5-4% NaTDCs, 0.1-1% dodecanes to take off cell solution described in step (7)
One kind of base sodium sulphate, 0.1-1%Triton X-100, alkyl glycosides, 0.05-5 μ g/ml trypsase or pepsin, step
Suddenly the de- cell solution selected in (7) is different one kind from the de- cell solution selected in step 5.
It is further preferred that described in step (9) protect liquid be physiological buffer, cell culture fluid, tissue preserration liquid, thoroughly
The one or more of bright matter acid solution, chitosan solution, dextran solution, glycerine etc..
It is further preferred that according to clinical demand, machine-shaping mode described in step (9) includes hygrometric state method or dry state
Method：Hygrometric state method is to sterilize after the de- bionical matrix of cell black carp skin of infiltration protection liquid is directly packed, sealed；Dry state rule will soak
The de- bionical matrix of cell black carp skin of profit protection liquid is first freeze-dried, then is packed, seals, sterilized.
It is bionical with cell black carp skin is taken off that the second aspect of the invention is to provide a kind of Regeneration and Repair prepared by the method
Matrix.
The third aspect of the invention is to provide a kind of Regeneration and Repair with taking off the bionical matrix of cell black carp skin again
The raw application repaired in a variety of soft tissues such as skin, tendon, peritonaeum, ligament, endocranium.
Compared with prior art, the Regeneration and Repair provided by the invention preparation method tool for taking off the bionical matrix of cell black carp skin
There is following technique effect：
(1) the abundant aquatic products processing waste black carp skin of china natural resources is efficiently used, it is imitative to prepare de- cell through special processing
Raw matrix is used for the Regeneration and Repair of tissue injury, can not only avoid the viral transmission of terricole source property acellular matrix material
Risk, and religion barrier can be evaded；
(2) the de- bionical matrix of cell black carp skin prepared by has been effectively retained native three dimensional structure, and cell removal efficiency is high；It should
The dry state and hygrometric state mechanical strength of bionical matrix are higher, can meet the requirement of clinical different operation；
(3) preparation process provided does not introduce organic solvent, and effective control has also been carried out to pyrogen and microorganism, can
Biological safety and compatibility are effectively improved, clinical application is convenient for.
Description of the drawings
Fig. 1 is a kind of Regeneration and Repair of present invention preparation technology flow chart for taking off the bionical matrix of cell black carp skin；
Fig. 2 is the Regeneration and Repair of the present invention apparent picture for taking off the bionical matrix of cell black carp skin；
Fig. 3 is the Regeneration and Repair of the present invention scanning electron microscopic picture for taking off the bionical matrix of cell black carp skin, shows the day of order
Right porous stent structure；
Fig. 4 is that Regeneration and Repair of the present invention dyes picture, substantially acellular residual with the HE for taking off the bionical matrix of cell black carp skin；
Fig. 5 is the Regeneration and Repair of the present invention hydrophilicity experiment picture for taking off the bionical matrix of cell black carp skin, shows hydrophily
Well.
Specific implementation mode
Process flow chart as shown in Figure 1, the object of the present invention is to provide a kind of de- cell black carp skins of Regeneration and Repair
The preparation method of bionical matrix, main technical schemes are formed using black carp skin as raw material is specially treated；The Regeneration and Repair
Include raw material pre-treatment with the de- bionical Matrix formulation procedure of cell black carp skin, thick arrangement, degreasing, de- cell, arrange again, preserve liquid
The processing steps such as pretreatment, molding.For Regeneration and Repair prepared by the present invention with the bionical matrix of cell black carp skin is taken off, cell removing is thorough
Bottom, natural porous network structure are complete, are the bionic extracellular matrix timbering materials of low immunogenicity, can be regeneration and restoration
Good microenvironment is provided with cell growth.Compared to the acellular matrix product in the sources such as common pig, ox, prepared by the present invention
The de- bionical matrix zoonotic virus of cell black carp skin carry and propagate risk is low, religious ethics risk is low, it is resourceful and at
This is cheap.
The present invention is described in more detail below by specific embodiment, for a better understanding of the present invention,
But following embodiments are not intended to limit the scope of the invention.
The preparation for taking off the bionical matrix of cell black carp skin of 1 Regeneration and Repair of embodiment
(1) black carp skin pre-processes：The tissues such as fresh black carp skin scraping off residual scale and redundant muscular, 4 DEG C of deionized waters are more
Secondary flushing eliminates residual blood；The fritter that 30 × 50mm is cut into according to different anatomic position is immediately placed in -40 DEG C of freezings, makes
With preceding taking-up, flowing water thaws at room temperature；
(2) mucous membrane/liquid and degreasing are removed：At 4 DEG C, with solid-liquid ratio 1:The 3.5%NaCl solution of 20 (w/w) stirs 1h heel row
Except waste liquid, thick arrange is carried out to black carp skin and removes partial redundance；Above step is repeated 3 times；
(3) micro-structure is protected：At 4 DEG C, with solid-liquid ratio 1:After PBS buffer solution (0.1M, pH7.4) the stirring 2h of 20 (w/w)
Exclude waste liquid；Above step is repeated 2 times；
(4) it cleans：At 4 DEG C, with solid-liquid ratio 1:The 3.5%NaCl solution stirring 4h heel row of 20 (w/w) removes waste liquid；
(5) de- cell processing for the first time：At 4 DEG C, with solid-liquid ratio 1:(sulfur acid celebrating is big mould for 1% NaTDC of 20 (w/w)
50 μ g/ml of element) stir process 12h, black carp skin is arranged again and removes the partial redundance tissue that above-mentioned processing procedure is formed；4
At DEG C, with solid-liquid ratio 1:Hank ' the s buffer solution for cleaning of 20 (w/w) 3 times, each 0.5h；
(6) micro-structure is protected again：At 4 DEG C, with solid-liquid ratio 1:The PBS buffer solution (0.1M, pH7.4) of 20 (w/w) stirs 2h
Heel row removes waste liquid；Above step is repeated 2 times；
(7) secondary de- cell processing：At 4 DEG C, with solid-liquid ratio 1:0.5% sodium dodecyl sulfate solution of 20 (w/w) (contains
50 μ g/ml of gentamicin sulphate) stir process 12h；At 4 DEG C, with solid-liquid ratio 1:Hank ' the s buffer solution for cleaning of 20 (w/w) 3 times,
Each 0.5h；
(8) it cleans：At 4 DEG C, with solid-liquid ratio 1:The 3.5%NaCl solution stirring 4h heel row of 20 (w/w) removes waste liquid；
(9) pretreatment of protection liquid and machine-shaping：At 4 DEG C, the de- bionical matrix of cell black carp skin of preparation is soaked in material
Liquor ratio 1:In 0.5% sodium hyaluronate solution of 20 (w/w), it is slowly stirred 2h, is freeze-dried, then pack, seal；
(10) final sterilization obtains finished product：The dry state of gained Packing Sound is taken off into the bionical matrix irradiation sterilization of cell black carp skin,
Finished product can be obtained.
The preparation for taking off the bionical matrix of cell black carp skin of 2 Regeneration and Repair of embodiment
(1) black carp skin pre-processes：With embodiment 1.
(2) mucous membrane/liquid and degreasing are removed：At 4 DEG C, with solid-liquid ratio 1:The 5%NaCl solution stirring 1h heel row of 10 (w/w) is removed
Waste liquid carries out thick arrange to black carp skin and removes partial redundance；Above step is repeated 2 times.
(3) micro-structure is protected：At 4 DEG C, with solid-liquid ratio 1:After PBS buffer solution (0.2M, pH7.0) the stirring 2h of 10 (w/w)
Exclude waste liquid；Above step is repeated 1 times.
(4) it cleans：At 4 DEG C, with solid-liquid ratio 1:The 5%NaCl solution stirring 4h heel row of 10 (w/w) removes waste liquid.
(5) de- cell processing for the first time：At 4 DEG C, with solid-liquid ratio 1:(sulfur acid celebrating is big mould for 2% NaTDC of 10 (w/w)
50 μ g/ml of element) stir process 18h, black carp skin is arranged again and removes the partial redundance tissue that above-mentioned processing procedure is formed；4
At DEG C, with solid-liquid ratio 1:Hank ' the s buffer solution for cleaning of 10 (w/w) 3 times, each 1h.
(6) micro-structure is protected again：At 4 DEG C, with solid-liquid ratio 1:The PBS buffer solution (0.2M, pH7.0) of 10 (w/w) stirs 2h
Heel row removes waste liquid；Above step is repeated 1 times.
(7) secondary de- cell processing：At 20 DEG C, with solid-liquid ratio 1:10 (w/w) trypsin solution (0.5 μ g/ml, with
PH8.0Tric-HCl buffer solution systems are prepared, 50 μ g/ml of sulfur acid gentamicin), stir process 18h；At 4 DEG C, with solid-liquid ratio
1:Hank ' the s buffer solution for cleaning of 10 (w/w) 3 times, each 1h.
(8) it cleans：At 4 DEG C, with solid-liquid ratio 1:The 5%NaCl solution stirring 4h heel row of 10 (w/w) removes waste liquid.
(9) pretreatment of protection liquid and machine-shaping：At 4 DEG C, the de- bionical matrix of cell black carp skin of preparation is soaked in material
Liquor ratio 1:In 0.5% sodium hyaluronate solution of 10 (w/w), it is slowly stirred 4h, pack, sealing.
(10) final sterilization obtains finished product：The hygrometric state of gained Packing Sound is taken off into the bionical matrix irradiation sterilization of cell black carp skin,
Finished product can be obtained.
The preparation for taking off the bionical matrix of cell black carp skin of 3 Regeneration and Repair of embodiment
(1) black carp skin pre-processes：With embodiment 1；
(2) mucous membrane/liquid and degreasing are removed：With embodiment 2；
(3) micro-structure is protected：At 4 DEG C, with solid-liquid ratio 1:After D-Hank ' s buffer solutions (pH7.2) the stirring 2h of 10 (w/w)
Exclude waste liquid；Above step is repeated 2 times.
(4) it cleans：At 4 DEG C, with solid-liquid ratio 1:The 4%NaCl solution stirring 4h heel row of 10 (w/w) removes waste liquid；
(5) de- cell processing for the first time：At 4 DEG C, with solid-liquid ratio 1:0.5% dodecyl sodium sulfate (the sulfur acid of 10 (w/w)
50 μ g/ml of gentamicin) stir process 12h, black carp skin is arranged again and removes the partial redundance that above-mentioned processing procedure is formed
Tissue；At 4 DEG C, with solid-liquid ratio 1:Hank ' the s buffer solution for cleaning of 10 (w/w) 3 times, each 1h；
(6) micro-structure is protected again：At 4 DEG C, with solid-liquid ratio 1:D-Hank ' the s buffer solutions (pH7.2) of 10 (w/w) stir 2h
Heel row removes waste liquid；Above step is repeated 2 times；
(7) secondary de- cell processing：With embodiment 2；
(8) it cleans：At 4 DEG C, with solid-liquid ratio 1:The 4%NaCl solution stirring 4h heel row of 10 (w/w) removes waste liquid；
(9) pretreatment of protection liquid and machine-shaping：At 4 DEG C, the de- bionical matrix of cell black carp skin of preparation is soaked in material
Liquor ratio 1:In the DMEM cell culture fluids of 10 (w/w), it is slowly stirred 4h, pack, sealing；
(10) final sterilization obtains finished product：With embodiment 2.
The preparation for taking off the bionical matrix of cell black carp skin of 4 Regeneration and Repair of embodiment
(1) black carp skin pre-processes：With embodiment 1.
(2) mucous membrane/liquid and degreasing are removed：At 4 DEG C, with solid-liquid ratio 1:The 5%NaCl solution stirring 1h heel row of 8 (w/w) is removed
Waste liquid carries out thick arrange to black carp skin and removes partial redundance；Above step is repeated 3 times.
(3) micro-structure is protected：At 4 DEG C, with solid-liquid ratio 1:The PBS buffer solution (0.2M, pH7.0) of 8 (w/w) stirs 1h heel row
Except waste liquid；Above step is repeated 2 times.
(4) it cleans：At 4 DEG C, with solid-liquid ratio 1:The 5%NaCl solution stirring 4h heel row of 8 (w/w) removes waste liquid.
(5) de- cell processing for the first time：At 4 DEG C, with solid-liquid ratio 1:(sulfur acid celebrating is big mould for 4% NaTDC of 8 (w/w)
50 μ g/ml of element) stir process 12h, black carp skin is arranged again and removes the partial redundance tissue that above-mentioned processing procedure is formed；4
At DEG C, with solid-liquid ratio 1:Hank ' the s buffer solution for cleaning of 8 (w/w) 3 times, each 1h.
(6) micro-structure is protected again：At 4 DEG C, with solid-liquid ratio 1:After PBS buffer solution (0.2M, pH7.0) the stirring 1h of 8 (w/w)
Exclude waste liquid；Above step is repeated 2 times.
(7) secondary de- cell processing：At 4 DEG C, with solid-liquid ratio 1:The 1%Triton X-100 solution (sulfur acids of 8 (w/w)
50 μ g/ml of gentamicin), stir process 18h；At 4 DEG C, with solid-liquid ratio 1:Hank ' the s buffer solution for cleaning of 8 (w/w) 3 times, every time
1h。
(8) it cleans：At 4 DEG C, with solid-liquid ratio 1:The 5%NaCl solution stirring 4h heel row of 8 (w/w) removes waste liquid.
(9) pretreatment of protection liquid and machine-shaping：At 4 DEG C, the de- bionical matrix of cell black carp skin of preparation is soaked in material
Liquor ratio 1:In Hank ' the s buffer solutions of 8 (w/w), it is slowly stirred 2h, is freeze-dried, pack, sealing.
(10) final sterilization obtains finished product：Electron beam irradiation sterilization obtains final finished.
The preparation for taking off the bionical matrix of cell black carp skin of 5 Regeneration and Repair of embodiment
1) black carp skin pre-processes：With embodiment 1.
2) mucous membrane/liquid and degreasing are removed：With embodiment 4.
3) micro-structure is protected：With embodiment 4.
4) it cleans：With embodiment 4.
5) de- cell processing for the first time：At 4 DEG C, with solid-liquid ratio 1:- 0.1% alkyl glycosides of 2% NaTDC of 8 (w/w)
(50 μ g/ml of sulfur acid gentamicin) stir process 18h, remaining is the same as embodiment 4.
6) micro-structure is protected again：With embodiment 4.
7) secondary de- cell processing：Solid-liquid ratio 1：8, remaining is the same as embodiment 2.
8) it cleans：With embodiment 4.
9) pretreatment of protection liquid and machine-shaping：At 4 DEG C, the de- bionical matrix of cell black carp skin of preparation is soaked in feed liquid
Than 1:In the glycerine of 8 (w/w), it is slowly stirred 4h, pack, sealing.
10) final sterilization obtains finished product：Irradiation sterilization obtains final finished.
Regeneration and Repair made from above-described embodiment 1 is scanned Electronic Speculum observation with de- cell black carp skin Biomimic materials
Judge that structure and cell slough situation after three-dimensional structure, histotomy and HE dyeing, and is wanted according to GB/T16886 relevant criterions
It asks and its Water absorption, hydrophily, expansion rate of water absorption, tearing strength and permeability etc. is detected.
If the testing result of Fig. 2-5 is shown, the prepared de- bionical matrix of cell black carp skin has natural bionic three-dimensional
Porous structure, moving into and being proliferated convenient for cell, de- cell black carp skin prepared by this method is almost without cell residue, immunogenicity
It is low.Water absorption is about 5 or so, and expansion rate of water absorption is about 9 or so, and hydrophily is good, can effectively be inhaled as wound repair material
Surface of a wound blood or body fluid are received, hydrophilic interface is convenient for the adherency and regeneration of cell.Tearing strength is up to 30MPa, after water suction
Material is not curled, and form still keeps regular, can meet the high soft tissue repair of mechanical strength requirement such as hernia paster, gynaecology's suspender belt
Deng.Permeability experimental result confirms that the bionical matrix liquid permeability of de- cell black carp skin prepared by this method is up to 8mS/cm, tool
There is good permeability, the Regeneration and Repair for wound tissue can provide good liquid-nutrition exchange media, also allow for the surface of a wound
The gas-liquid at place exchanges, and can effectively avoid the infection because liquid agglutination causes below material or inflammation risk.Above-mentioned testing result table
Bright, the de- bionical matrix of cell black carp skin prepared by the present invention is a kind of ideal regeneration and restoration material and natural scaffold material
Material, has huge applications potentiality in regeneration and restoration field.
The present invention respectively by obtained by above-mentioned other embodiment Regeneration and Repair with take off cell black carp skin Biomimic materials into
The above-mentioned coherent detection of row, acquired results are similar to Application Example 1.
Specific embodiments of the present invention are described in detail above, but it is intended only as example, the present invention is simultaneously unlimited
It is formed on particular embodiments described above.To those skilled in the art, it is any to the equivalent modifications that carry out of the present invention and
It substitutes also all among scope of the invention.Therefore, without departing from the spirit and scope of the invention made by impartial conversion and
Modification, all should be contained within the scope of the invention.
Claims (10)
1. a kind of Regeneration and Repair preparation method for taking off the bionical matrix of cell black carp skin, which is characterized in that using black carp skin as raw material,
The Regeneration and Repair bionical matrix of de- cell black carp skin is made after the processing such as degreasing, de- cell.
2. the Regeneration and Repair according to claim 1 preparation method for taking off the bionical matrix of cell black carp skin, which is characterized in that
Include the following steps：
(1) black carp skin pre-processes：By the cleaning of fresh black carp skin, removal redundancy tissue and impurity；
(2) mucous membrane/liquid and degreasing are removed：At 4 DEG C, step (1) treated black carp skin carries out thick arrange and removes partial redundance；
(3) micro-structure is protected：At 4 DEG C, with solid-liquid ratio 1:5-1:Phosphate buffer (0.05-0.2M, the pH7.0- of 20 (w/w)
7.4) stirring 2-4h heel row removes waste liquid；Above step repeats 1-2 times.
(4) it cleans：At 4 DEG C, with solid-liquid ratio 1:5-1:The 2%-5%NaCl solution stirring 4h heel row of 20 (w/w) removes waste liquid.
(5) de- cell processing for the first time：At 4 DEG C, with solid-liquid ratio 1:5-1:The de- cell solution stir process 12-20h of 20 (w/w),
Black carp skin is arranged again and removes the partial redundance tissue that above-mentioned processing procedure is formed；At 4 DEG C, with solid-liquid ratio 1:5-1:20
(w/w) physiological buffer cleans 2-4 times, each 0.5-4h.
(6) micro-structure is protected again：At 4 DEG C, with solid-liquid ratio 1:5-1:20 (w/w) phosphate buffer (0.05-0.2M,
PH7.0-7.4) stirring 2-4h heel row removes waste liquid；Above step repeats 1-2 times.
(7) secondary de- cell processing：Selection handles different de- cell systems and corresponding reaction condition from first de- cell,
Carry out secondary de- cell processing, solid-liquid ratio 1:5-1:20(w/w).
(8) it cleans：At 4 DEG C, with solid-liquid ratio 1:5-1:The 2%-5%NaCl solution stirring 4h heel row of 20 (w/w) removes waste liquid.
(9) pretreatment of protection liquid and machine-shaping：At 4 DEG C, the de- bionical matrix of cell black carp skin of preparation is soaked in solid-liquid ratio
1:5-1:In the protection liquid of 20 (w/w), it is slowly stirred 2-8h；And machine-shaping, the bionical matrix of de- cell black carp skin, packet is made
Dress；
(10) final sterilization obtains finished product：The dry state of gained Packing Sound or hygrometric state are taken off the bionical matrix of cell black carp skin finally to go out
Bacterium can obtain finished product.
3. the Regeneration and Repair according to claim 2 preparation method for taking off the bionical matrix of cell black carp skin, which is characterized in that
The black carp skin is the material of fresh stripping, and there is trackability in source and meets quarantine request.
4. the Regeneration and Repair according to claim 2 preparation method for taking off the bionical matrix of cell black carp skin, which is characterized in that
The pretreated specific treatment process of black carp skin is：By tissues such as fresh black carp skin scraping off residual scale and redundant musculars, 4 DEG C
Deionized water repeatedly rinses and eliminates residual blood；And suitable dimension is cut into according to different anatomic position, be immediately placed in -40 DEG C it is cold
Freeze, using preceding taking-up, flowing water thaws at room temperature.
5. the Regeneration and Repair according to claim 2 preparation method for taking off the bionical matrix of cell black carp skin, which is characterized in that
Removal mucous membrane/the liquid and the concrete technology of degreasing are：At 4 DEG C, with solid-liquid ratio 1:5-1:The 2%-5%NaCl of 20 (w/w) is molten
Liquid stirs 1-4h heel row and removes waste liquid, and carrying out thick arrange to black carp skin removes partial redundance, and above step repeats 2-4 times.
6. the Regeneration and Repair according to claim 2 preparation method for taking off the bionical matrix of cell black carp skin, which is characterized in that
It is 0.5-4% NaTDCs, 0.1-1% lauryl sodium sulfate, 0.1-1% that cell solution is taken off described in step (5)
One kind of Triton X-100, alkyl glycosides.
7. the Regeneration and Repair according to claim 2 preparation method for taking off the bionical matrix of cell black carp skin, which is characterized in that
It is 0.5-4% NaTDCs, 0.1-1% lauryl sodium sulfate, 0.1-1% that cell solution is taken off described in step (7)
One kind of Triton X-100, alkyl glycosides, 0.05-5 μ g/ml trypsase or pepsin.
8. the Regeneration and Repair according to claim 2 preparation method for taking off the bionical matrix of cell black carp skin, which is characterized in that
Protect liquid molten for physiological buffer, cell culture fluid, tissue preserration liquid, hyaluronic acid solution, chitosan described in step (9)
The one or more of liquid, dextran solution, glycerine etc..
9. a kind of Regeneration and Repair bionical base of de- cell black carp skin such as prepared according to any one of claim 1-8 the methods
Matter.
10. a kind of Regeneration and Repair as claimed in claim 9 is with the de- bionical matrix of cell black carp skin in Regeneration and Repair skin, flesh
Application in a variety of soft tissues such as tendon, peritonaeum, ligament, endocranium.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810344297.1A CN108478869A (en) | 2018-04-17 | 2018-04-17 | A kind of Regeneration and Repair bionical matrix of de- cell black carp skin and its preparation method and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810344297.1A CN108478869A (en) | 2018-04-17 | 2018-04-17 | A kind of Regeneration and Repair bionical matrix of de- cell black carp skin and its preparation method and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108478869A true CN108478869A (en) | 2018-09-04 |
Family
ID=63316311
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810344297.1A Pending CN108478869A (en) | 2018-04-17 | 2018-04-17 | A kind of Regeneration and Repair bionical matrix of de- cell black carp skin and its preparation method and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108478869A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111359016A (en) * | 2020-03-13 | 2020-07-03 | 青岛海洋生物医药研究院 | Low-antigenicity fish skin tissue and processing method thereof |
| CN112426566A (en) * | 2020-11-30 | 2021-03-02 | 山东大学 | Regenerative cornea material and preparation method and application thereof |
| CN114949330A (en) * | 2022-06-16 | 2022-08-30 | 国科温州研究院(温州生物材料与工程研究所) | Acellular fish skin matrix and preparation method thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4970298A (en) * | 1984-03-27 | 1990-11-13 | University Of Medicine And Dentistry Of New Jersey | Biodegradable matrix and methods for producing same |
| CN1096458A (en) * | 1993-11-25 | 1994-12-21 | 徐国士 | The fish skin of treatment burn (knife injury) wound surface (wet or dried) xenogenesis skin and manufacturing process |
| CN102781485A (en) * | 2009-10-07 | 2012-11-14 | 克雷西斯公司 | A scaffold material for wound care and/or other tissue healing applications |
| CN103320485A (en) * | 2013-06-03 | 2013-09-25 | 王南平 | Preparation method of fish-skin collagen for medical biomaterial |
| WO2013144727A2 (en) * | 2012-03-30 | 2013-10-03 | Kerecis Ehf | A scaffold material graft for wound care and/or other tissue healing applications |
| CN106075579A (en) * | 2015-03-31 | 2016-11-09 | 柏登生医股份有限公司 | Fish skin derived tissues repair materials and manufacture method thereof |
-
2018
- 2018-04-17 CN CN201810344297.1A patent/CN108478869A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4970298A (en) * | 1984-03-27 | 1990-11-13 | University Of Medicine And Dentistry Of New Jersey | Biodegradable matrix and methods for producing same |
| CN1096458A (en) * | 1993-11-25 | 1994-12-21 | 徐国士 | The fish skin of treatment burn (knife injury) wound surface (wet or dried) xenogenesis skin and manufacturing process |
| CN102781485A (en) * | 2009-10-07 | 2012-11-14 | 克雷西斯公司 | A scaffold material for wound care and/or other tissue healing applications |
| WO2013144727A2 (en) * | 2012-03-30 | 2013-10-03 | Kerecis Ehf | A scaffold material graft for wound care and/or other tissue healing applications |
| CN103320485A (en) * | 2013-06-03 | 2013-09-25 | 王南平 | Preparation method of fish-skin collagen for medical biomaterial |
| CN106075579A (en) * | 2015-03-31 | 2016-11-09 | 柏登生医股份有限公司 | Fish skin derived tissues repair materials and manufacture method thereof |
Non-Patent Citations (3)
| Title |
|---|
| SKULI MAGNUSSON 等: "Regenerative and Antibacterial Properties of Acellular Fish Skin Grafts and Human Amnion/Chorion Membrane: Implications for Tissue Preservation in Combat Casualty Care", 《MILITARY MEDICINE》 * |
| 位晓娟 等: "脱细胞鱼皮基质作为新型组织工程支架的研究进展", 《中国修复重建外科杂志》 * |
| 夏凯 等: "芒鲶胶原组织再生膜的制备及骨修复作用研究", 《中国海洋药物》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111359016A (en) * | 2020-03-13 | 2020-07-03 | 青岛海洋生物医药研究院 | Low-antigenicity fish skin tissue and processing method thereof |
| CN112426566A (en) * | 2020-11-30 | 2021-03-02 | 山东大学 | Regenerative cornea material and preparation method and application thereof |
| CN114949330A (en) * | 2022-06-16 | 2022-08-30 | 国科温州研究院(温州生物材料与工程研究所) | Acellular fish skin matrix and preparation method thereof |
| CN114949330B (en) * | 2022-06-16 | 2024-02-20 | 国科温州研究院(温州生物材料与工程研究所) | Acellular fishskin matrix and preparation method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP5993387B2 (en) | Collagen biofiber and its preparation method and use | |
| CN108355172A (en) | A kind of soft tissue repair bionical matrix of de- cell Java tilapia skin and its preparation method and application | |
| JP6254094B2 (en) | Cross-linked dehydrated placental tissue graft and methods of making and using the same | |
| CN108478869A (en) | A kind of Regeneration and Repair bionical matrix of de- cell black carp skin and its preparation method and application | |
| CA2826433A1 (en) | Micronized placental tissue compositions and methods of making and using the same | |
| CN101366979B (en) | Tissue patch and preparation method thereof | |
| CN105797212A (en) | Preparing method and application of acellular amniotic membrane used for repairing skin wound difficult to heal | |
| KR20130118418A (en) | Acellular treatment for material derived from biological tissue, a method for treating using thereof, and biological material obtained from this method | |
| CN112618799B (en) | Fish skin acellular dermal matrix and preparation method and application thereof | |
| CN107233621A (en) | Natural soft tissue goes the preparation method of cellular matrix | |
| CN110193096A (en) | A kind of marine origin biomimetic type cartilage material and preparation method thereof | |
| CN110051884A (en) | A kind of bio-sponge and preparation method based on Acelluar small intestinal submucosa | |
| CN103961752B (en) | Tissue regeneration guiding film and preparation method thereof | |
| CN111084900A (en) | Preparation method and application of acellular fish skin matrix | |
| CN109364298A (en) | A kind of preparation method of acellular dermal matrix material | |
| CN102772822A (en) | Application of collagen matrix as tissue engineering scaffold | |
| CN109078222A (en) | A kind of novel fish-skin source oral restoration film and preparation method thereof | |
| CN107412868A (en) | A kind of preparation method of acellular dermal matrix and obtained acellular dermal matrix | |
| RU2353397C2 (en) | Bioabsorbable collagen matrix, method of production and application | |
| CN105079881B (en) | A kind of vagina host material and preparation method thereof | |
| Kumaresan et al. | Development of Human Umbilical cord based scaffold for tissue engineering application | |
| CN109589455A (en) | A kind of thermal denaturation collagen or thermal denaturation dermis scaffold substitute and preparation method | |
| Chai et al. | Applications of Decellularized Extracellular Matrix for Regenerative Medicine | |
| WO2021128763A1 (en) | Preparation method for placenta tissue engineering de-immunized skin scaffold | |
| CN112494724A (en) | Medical repairing periosteum and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |