CN105833353A - Preparation and application of bioengineering decellularized dermal matrix - Google Patents

Preparation and application of bioengineering decellularized dermal matrix Download PDF

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Publication number
CN105833353A
CN105833353A CN201610297083.4A CN201610297083A CN105833353A CN 105833353 A CN105833353 A CN 105833353A CN 201610297083 A CN201610297083 A CN 201610297083A CN 105833353 A CN105833353 A CN 105833353A
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preparation
skin
dermal matrix
detergent
concentration
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CN105833353B (en
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史真
史伟云
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Baiodisel (Chengdu) Biotechnology Co.,Ltd.
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Diesel (beijing) Biological Technology Co Ltd
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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
    • A61L27/50—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
    • 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
    • A61L27/36—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
    • 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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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Dermatology (AREA)
  • Medicinal Chemistry (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Transplantation (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Botany (AREA)
  • Molecular Biology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Zoology (AREA)
  • Materials For Medical Uses (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Detergent Compositions (AREA)

Abstract

The invention discloses a novel bioengineering decellularized dermal matrix and a preparing method thereof. The preparing method includes the steps that porcine skin is pretreated to obtain dermal layer skin graft, and a mixed protective solution is used in all the dermal matrix decellularization process; a high hydrostatic pressure technology is used for preseparating cells in dermal tissue; residual cell nucleuses are digested through a composite nuclease method; detergent is used for removing loose smudge cell sheets; the mixed protective solution is used for rinsing. Suitable and efficient physical means are used and supplemented with a small amount of enzymes and detergent, cell components in the dermis are fully removed, under whole-process protection of the protective solution, it can be ensured that immunogenicity of the dermal matrix is reduced to the maximum on the basis of ensuring complete removal of the cell nucleuses, a normal three-dimensional collagenous fiber structure and array polarity of the dermal matrix are reserved, and therefore the prepared decellularized dermal matrix is high in stability and biocompatibility.

Description

The preparation of a kind of bioengineering acellular dermal matrix and purposes
Technical field
The present invention relates to the cell free preparation method of the preparation field of bioengineering medical material, specially dermal matrix
Background technology
There is the huge surface of a wound of skin deficiencies or difficult healing in the patients such as severe trauma, large-area burns and skin ulcer, and treats and lack The surface of a wound damaged, substantially based on self-skin transplant, but the most autologous skin source is nervous, especially obtains when large skin defect Take from body skin the most increasingly difficult.On the other hand, taking autologous skin is the wound new to patient；So at the therapeutic process of these diseases In, use Graftskin temporary transient or long-term flap coverage, become clinical needs.
Natural leather material is due to similar to human skin, therefore becomes dermatoplastic first-selection.Wherein xenogenesis skin is cheap, comes Source is extensive, but there is variant cell antigenicity, it is easy to strong immunological rejection occurs, and eliminating its antigenicity just can become reason The Graftskin thought.The most how xenogeneic skin is taken off cell, remove xenogeneic skin antigenicity, protect its complete corium simultaneously The three-dimensional structure of collagenous fibres, becomes the focus that current tissue engineered dermal equivalent is developed.
Existing acellular dermal manufacturing technology is the method for chemical reagent process and destroys its cell, and combines the crosslinking agents such as glutaraldehyde Crosslinking reduces immunogenicity, there is destruction collagen structure, leaves over the shortcomings such as bio-toxicity.It is also proposed employing physics in recent years Means removing xenogenesis chrotoplast method, it is desirable to solve chemical reagent processing method prepare leather material exist shortcoming, but this A little method for removing cells frequently can lead to tissue immanent structure and destroyed, and lose the original basic biological function of de-cell tissue.
Therefore for the deficiencies in the prior art, it is provided that a kind of immunogenicity that can either reduce dermal matrix, dermal matrix is retained again Acellular dermal matrix of normal collagen structure and preparation method thereof is the task of top priority.
Summary of the invention
The invention provides a kind of omnidistance use special protection liquid and suitable efficient physics compound biological enzyme obtains acellular dermal The method of matrix.
It is at the innovation of the present invention: 1, the skin to pig obtains skin corium skin graft after pre-processing；2, dermal matrix is de-thin During born of the same parents, omnidistance use comprises the hybrid protection liquid of one or more dermis protective ingredients；3, high static pressure technology is used to divide in advance Cell in skin histology；4, composite nucleic acid enzyme method digestion residual cells core is used；5, utilize detergent removing loose broken Broken cell sheet；6, hybrid protection liquid is used to rinse.De-cell step sets suitable temperature, time and detergent, disappears Change enzyme concentration conjunctival tissue is processed.
The concentration that consumption is chondroitin sulfate of various protectiveness compositions is 30-80g/L；The concentration of hyaluronic acid is 20-50g/L； The concentration of glycerine is 100-500g/L；Neomycin 5-10mg/L.
High static pressure pressure condition is 400-600MPa, and high static pressure frequency is 5～10 times, is 2～5 minutes every time.
The concentration of DNA enzymatic is 2000-4000U/ml, and the concentration of nuclease is 2000-4000U/ml.DNA enzymatic or nuclease The de-nucleus time is 3-6 hour, and treatment temperature is 15-30 degree.
Detergent condition is concentration 0.1-5% of Pluronic F68, concentration 0.1-5% of sarcosyl, PEG 400 Concentration 5-10%.The detergent process time is 3-6 hour.
The most simultaneously or separately carry out the process of enzyme and detergent.
Accompanying drawing explanation
Fig. 1 is the HE colored graph of normal pig skin biopsy
Fig. 2 is the DAPI colored graph of de-cell porcine skin section
Fig. 3 is the result that skin histology takes off cell whole process use protection liquid
Fig. 4 is that skin histology takes off the omnidistance result not using protection liquid of cell
Fig. 5 is that skin histology takes off the result using detergent in cell processes
Fig. 6 is that skin histology takes off the result not using detergent in cell processes
Fig. 7 is that skin histology takes off the result using digestive ferment in cell processes
Fig. 8 is that skin histology takes off the result not using digestive ferment in cell processes
Detailed description of the invention
Being specifically described the present invention below by embodiment, these embodiments are served only for being described in further detail the explanation present invention, It is not intended that limiting the scope of the present invention, within scope, make nonessential adjustment need side of the present invention Authorize.
Embodiments of the invention 1
Taking pig back skin, after cleaned, unhairing, leaving and taking thickness is 0.3-0.5mm, and width is that the middle thick corium skin graft of 1-5cm is standby With.
1, omnidistance use protects liquid to protect dermal tissue, and protection formula of liquid is: RPMI-1640 culture medium, adds sulfuric acid Chondroitin 40g/L, hyaluronic acid 20g/L, neomycin 5mg/L, adjust pH value to 7.2, and osmotic pressure is 380mOsm；
2, corium skin graft is sealed in the polybag filling protection liquid；
3, under 400MPa height hydrostatic pressure condition, processing 8 times, each time is 5 minutes；
4, after being taken out by corium, being placed in the protection liquid containing 2%Pluronic F68+2000U/ml DNA enzymatic, temperature is 30 DEG C, Set shaking table speed as 100 revs/min, process 4 hours；
5, after detergent and ferment treatment, take out conjunctiva and be placed in protection liquid and rinse 3 hours.
Embodiments of the invention 2
Taking pig back skin, after cleaned, unhairing, leaving and taking thickness is 0.3-0.5mm, and width is that the middle thick corium skin graft of 1-5cm is standby With.
1, omnidistance use protects liquid to protect eye conjunctival tissue, and protection formula of liquid is: RPMI-1640 culture medium, adds sulphur Aching and limp ossein 50g/L, glycerine 200g/L, neomycin 5mg/L, adjust pH value to 7.2, and osmotic pressure is 400mOsm；
2, corium skin graft is sealed in the polybag filling protection liquid；
3, under 600MPa height hydrostatic pressure condition, processing 5 times, each time is 3 minutes；
4, after being taken out by corium, being placed in the protection liquid containing 5% sarcosyl+4000U/ml DNA enzymatic, temperature is 25 DEG C, set shaking table speed as 100 revs/min, process 3 hours；
5, after detergent and ferment treatment, take out conjunctiva and be placed in protection liquid and rinse 6 hours.
Embodiments of the invention 3
Taking pig back skin, after cleaned, unhairing, leaving and taking thickness is 0.3-0.5mm, and width is that the middle thick corium skin graft of 1-5cm is standby With.
1, omnidistance use protects liquid to protect eye conjunctival tissue, and protection formula of liquid is: RPMI-1640 culture medium, adds sulphur Aching and limp ossein 30g/L, hyaluronic acid 50g/L, neomycin 5mg/L, adjust pH value to 7.2, and osmotic pressure is 450mOsm；
2, corium skin graft is sealed in the polybag filling protection liquid；
3, under 800MPa height hydrostatic pressure condition, processing 5 times, each time is 2 minutes；
4, after being taken out by corium, being placed in the protection liquid containing 2% dodecyl sodium sulfate+2000U/ml DNA enzymatic, temperature is 30 DEG C, set shaking table speed as 100 revs/min, process 5 hours；
5, after detergent and ferment treatment, take out conjunctiva and be placed in protection liquid and rinse 8 hours.
Following comparing result is all to be configured according to the de-cell step of embodiment 1.
Fig. 3 and Fig. 4 is that dermal tissue takes off cell whole process use protection liquid and the omnidistance results contrast not using protection liquid respectively.Not Protection liquid group whole process is used to use sterilized water for injection to replace protection liquid.By Fig. 3 and Fig. 4, it is seen that use protection liquid group de-thin Born of the same parents are complete, do not use protection liquid (sterilized water for injection) group to have obvious nucleus remaining.
Fig. 5 and Fig. 6 is to use detergent and do not use the results contrast of detergent respectively.As shown in Figure 5 and Figure 6, make to spend It is complete that dirt agent group takes off cell, and does not uses detergent group to have obvious nucleus remaining.
Fig. 7 and Fig. 8 is to use digestive ferment and do not use the results contrast of digestive ferment respectively.Visible use digestive ferment group cell is de-dry Only, digestive ferment group is not used to have a large amount of nucleus to colour.
The foregoing is only presently preferred embodiments of the present invention, not in order to limit the present invention, all spirit in the present invention and former Within then, any modification, equivalent substitution and improvement etc. made, should be included within the scope of the present invention.

Claims (11)

1. novel bioengineering acellular dermal matrix and preparation method thereof, specifically comprises the following steps that
(1) skin to pig obtains skin corium skin graft after pre-processing；
(2) use comprises one or more dermis protectant hybrid protection liquid；
(3) cell in high static pressure technology pre-separation dermal tissue is used；
(4) composite nucleic acid enzyme method digestion residual cells core is used；
(5) detergent is utilized to remove loose crushing cell sheet；
(6) carry out rinsing at dermal matrix hybrid protection liquid and remove unnecessary biology enzyme and cell residue；
(7) three-dimensional structure with normal collagenous fibres and the acellular dermal layer skin with biological nature arranging polarity are obtained Sheet.
2. preparation method as claimed in claim 1, takes pig back skin, and after cleaned unhairing, leaving and taking thickness is 0.3-0.5mm, width Middle thick corium skin graft for 1-5cm is standby.
3. preparation method as claimed in claim 1, hybrid protection liquid such as includes RPMI-1640 culture medium, chondroitin sulfate, thoroughly Bright matter acid, glycerine, neomycin etc. and combinations thereof, and ph value and infiltration need to be regulated be pressed onto Appropriate.
4. preparation method as claimed in claim 1, high static pressure technology includes different pressure, time and frequency parameters.
5. preparation method as claimed in claim 1, composite nucleic acid enzyme includes DNA enzymatic, RNase, other nucleases and group thereof Close.
6. preparation method as claimed in claim 1, detergent includes polyethylene glycol (PEG), Pluronic, dodecyl sulphate One or more in sodium, sarcosyl, natural gum, sodium alginate, cyclodextrin, CHAPS are as combination.
7. in preparation method, with dermal matrix hybrid protection liquid as claimed in claim 1, the time of rinsing is 3～8 hours.
8. preparation method as claimed in claim 3, Qi Gecheng is grouped into: RPMI-1640 culture medium contains, but not exclusively includes sulphur The concentration of aching and limp ossein is 30-80g/L；The concentration of hyaluronic acid is 10-50g/L；Glycerine 100-500g/L；Neomycin 5-10mg/L, regulation ph value is between 6.8-7.2, and osmotic pressure is 350-450mOsm.
9. preparation method as claimed in claim 4, described high static pressure pressure condition is 400-600MPa, and high static pressure frequency is 5～10 Secondary, it is 2～5 minutes every time；
10. preparation method as claimed in claim 5, described composite nucleic acid enzyme uses concentration during DNA enzymatic to be 2000-4000U/ml, use nuclease time the de-nucleus that concentration is 2000-4000U/ml, DNA enzymatic or nuclease time Between be 3-6 hour, treatment temperature is 15-30 DEG C.
11. preparation methods as claimed in claim 6, wherein said detergent Pluronic F68 concentration is 0.1-5%, dodecyl flesh The concentration of propylhomoserin sodium be the concentration of 0.1-5%, PEG 400 be 5-10%.Detergent uses the time to be 1-8 hour.
CN201610297083.4A 2016-05-09 2016-05-09 A kind of preparation of bioengineering acellular dermal matrix and purposes Active CN105833353B (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109550081A (en) * 2018-11-27 2019-04-02 中国人民解放军总医院第附属医院 A kind of de- antigen nerve processing method
CN111437443A (en) * 2020-04-20 2020-07-24 百澳瑞派（天津）生物科技有限公司 Novel dermal matrix acellular method
WO2021159198A1 (en) 2020-02-14 2021-08-19 Kheiros Pater Inovação S.A Method for producing decellularized biomaterial, decellularized biomaterial and use thereof

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030187515A1 (en) * 2002-03-26 2003-10-02 Hariri Robert J. Collagen biofabric and methods of preparing and using the collagen biofabric
CN101773687A (en) * 2009-12-31 2010-07-14 陕西瑞盛生物科技有限公司 Preparation method of composite soft-tissue patch
CN102470149A (en) * 2009-08-18 2012-05-23 生命细胞公司 Method for processing tissues
CN102573945A (en) * 2009-09-11 2012-07-11 翰林大学校产学协力团 Production method for cryopreserved acellular dermal matrix, and cryopreserved acellular dermal matrix produced thereby
CN103249404A (en) * 2010-07-02 2013-08-14 北卡罗来纳-查佩尔山大学 Biomatrix scaffolds

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030187515A1 (en) * 2002-03-26 2003-10-02 Hariri Robert J. Collagen biofabric and methods of preparing and using the collagen biofabric
CN102470149A (en) * 2009-08-18 2012-05-23 生命细胞公司 Method for processing tissues
CN102573945A (en) * 2009-09-11 2012-07-11 翰林大学校产学协力团 Production method for cryopreserved acellular dermal matrix, and cryopreserved acellular dermal matrix produced thereby
CN101773687A (en) * 2009-12-31 2010-07-14 陕西瑞盛生物科技有限公司 Preparation method of composite soft-tissue patch
CN103249404A (en) * 2010-07-02 2013-08-14 北卡罗来纳-查佩尔山大学 Biomatrix scaffolds

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109550081A (en) * 2018-11-27 2019-04-02 中国人民解放军总医院第附属医院 A kind of de- antigen nerve processing method
CN109550081B (en) * 2018-11-27 2021-07-16 中国人民解放军总医院第四医学中心 Antigen-removing nerve treatment method
WO2021159198A1 (en) 2020-02-14 2021-08-19 Kheiros Pater Inovação S.A Method for producing decellularized biomaterial, decellularized biomaterial and use thereof
CN111437443A (en) * 2020-04-20 2020-07-24 百澳瑞派（天津）生物科技有限公司 Novel dermal matrix acellular method

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