CN105833356B - A kind of new de- cell Valved allograft support and preparation method thereof - Google Patents
A kind of new de- cell Valved allograft support and preparation method thereof Download PDFInfo
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- CN105833356B CN105833356B CN201610297151.7A CN201610297151A CN105833356B CN 105833356 B CN105833356 B CN 105833356B CN 201610297151 A CN201610297151 A CN 201610297151A CN 105833356 B CN105833356 B CN 105833356B
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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
- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
- A61L31/005—Ingredients of undetermined constitution or reaction products thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- 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)
- Epidemiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Heart & Thoracic Surgery (AREA)
- Surgery (AREA)
- Vascular Medicine (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
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- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
Abstract
The invention discloses a kind of new bioengineering to take off cell Valved allograft support and preparation method thereof.Its invention main points includes：During whole use the hybrid protection liquid for including one or more Valved allograft structural defence agent；Using the cell in high static pressure technology pre-separation Valved allograft tissue；Using residual cells core in a small amount of composite nucleic acid enzyme and de-sludging agent method digestion Valved allograft tissue；Rinsed using hybrid protection liquid；The Valved allograft tissue of cell can be purified completely in 6 hours.The present invention can be on the basis of nucleus be de- totally, the immunogenicity in Valved allograft tissue is reduced to greatest extent, the three-dimensional structure and arrangement polarity of the normal collagenous fibres of Valved allograft tissue are remained simultaneously, make obtained de- cell Valved allograft tissue that there is good biocompatibility, and there is stronger biomechanical property.
Description
Technical field
The present invention relates to the preparation field of bioengineering medical material, the preparation side of specially de- cell Valved allograft support
Method.
Background technology
The treatment of the wound of complicated big vascular malformation and big blood vessel, particularly children and infant's Congenital Heart and blood vessel
The disease of deformity is, it is necessary to which the blood vessel or valved conduit of various diameters and size are repaired.The blood vessel someone clinically applied at present
Work synthesizes and biological blood vessel or the major class of valved conduit two.Synthetic material (or material modified), due to have be difficult to overcome it is non-
The problem of physiological and elasticity and pulling force, and normal blood flow state is hindered, it is high plus postoperative thrombus rate, it is necessary to eventually
Body takes anticoagulant possibility, so not being applied clinically.And preferably biological Valved allograft has natural branch
Frame structure, optimal haemodynamics, the advantages of being not required to anti-freezing, wherein more ripe with the clinical application technique of allogeneic blood vessel
And have preferable therapeutic effect, but China does not have brain death method, and valved homograft can not almost obtain.So seek different
The blood vessel of kind allosome turns into the focus and difficult point that Present clinical treats complicated department of cardiovascular surgery treatment.And heterogenous allosome is with valve at present
Blood vessel takes off cell and the technology of antigen there is a problem that clinical practice is in the starting stage.
The content of the invention
The invention provides a kind of quick, whole process to use special protection liquid and efficient physics compound biological enzyme means system
The method of standby de- cell Valved allograft timbering material.
The invention of the present invention is characterized by：1st, whole use includes one or more blood vessels in the de- cell processes of Valved allograft
The hybrid protection liquid of structural defence composition；2nd, using the cell in high static pressure technology pre-separation vascular tissue；3rd, using complex nucleus
Sour enzyme method digests residual cells core；4th, loose crushing cell sheet is removed using detergent；5th, floated using hybrid protection liquid
Wash.De- cell step sets suitable temperature, time and detergent, digestion enzyme concentration is handled intravascular stent.
The dosage of various protectiveness compositions is that the concentration of chondroitin sulfate is 20-50g/L；The concentration of dextran is 10-
20g/L；Glycerol concentration is 100-200g/L；Neomycin 5-10mg/L.
High static pressure pressure condition is 200-500MPa, and high static pressure frequency is 3~6 times, is every time 1~3 minute.
The concentration of DNA enzymatic is 1000-2000U/ml, and the concentration of nuclease is 1000-2000U/ml.DNA enzymatic or nuclease
The de- nucleus time be 2-4 hours, treatment temperature is 15-30 degrees Celsius.
Detergent condition is concentration 0.1-5%, the concentration 0.1-5% of lauryl sodium sulfate of dodecyl sodium sulfate,
The concentration 0.1-5% of sodium alginate.Detergent processing time is 2-4 hours.
Optionally simultaneously or separately carry out the processing of enzyme and detergent.
The Valved allograft support of preparation need to be attached on nitroglycerin mould, be put into anhydrous calcium chloride molecular sieve and be dehydrated, warp
Pack is sealed and marked, and the Gamma x ray irradiation xs for being 25kGy with irradiation dose save backup after sterilizing 5-10 hours at 4 DEG C.
Embodiment
The present invention is specifically described below by embodiment, these embodiments are served only for being described in further detail explanation originally
Invention, it is impossible to be interpreted as limiting the scope of the present invention, nonessential adjustment is made within the scope of the present invention to be needed
Present invention side authorizes.
Embodiments of the invention 1
The coronary artery for taking the aorta ascendens, the arch of aorta and 6-8mm of fresh adult pig or ox to grow is cleaned with PBS solution
Afterwards, it is put into the preservation of hybrid protection liquid.
1st, whole that Valved allograft support is protected using protection liquid, protection formula of liquid is：RPMI-1640 culture mediums,
Add chondroitin sulfate 50g/L, Dextran 10 g/L, neomycin 5mg/L, tune pH value to 7.2, osmotic pressure 300mOsm；
2nd, intravascular stent is sealed in the polybag for filling protection liquid；
3rd, under the conditions of 200MPa high static pressures, handle 6 times, each time is 3 minutes；
4th, after blood vessel is taken out, it is placed in the protection liquid containing 2% lauryl sodium sulfate+1000U/ml DNA enzymatics, temperature
For 30 DEG C, shaking table speed is set as 100 revs/min, is handled 2 hours；
5th, after detergent and ferment treatment, take out Valved allograft and be placed in protection liquid and rinse 3 hours.
Embodiments of the invention 2
The coronary artery for taking the aorta ascendens, the arch of aorta and 6-8mm of fresh adult pig or ox to grow is cleaned with PBS solution
Afterwards, it is put into the preservation of hybrid protection liquid.
1st, whole that Valved allograft support is protected using protection liquid, protection formula of liquid is：RPMI-1640 culture mediums,
Add chondroitin sulfate 20g/L, glycerine 100g/L, neomycin 5mg/L, tune pH value to 7.2, osmotic pressure 350mOsm；
2nd, intravascular stent is sealed in the polybag for filling protection liquid；
3rd, under the conditions of 400MPa high static pressures, handle 4 times, each time is 2 minutes；
4th, after blood vessel is taken out, it is placed in the protection liquid containing 0.5% sodium alginate+1500U/ml DNA enzymatics, temperature 25
DEG C, shaking table speed is set as 100 revs/min, is handled 4 hours；
5th, after detergent and ferment treatment, take out Valved allograft and be placed in protection liquid and rinse 2 hours.
Embodiments of the invention 3
The coronary artery for taking the aorta ascendens, the arch of aorta and 6-8mm of fresh adult pig or ox to grow is cleaned with PBS solution
Afterwards, it is put into the preservation of hybrid protection liquid.
1st, whole that Valved allograft support is protected using protection liquid, protection formula of liquid is：RPMI-1640 culture mediums,
Add chondroitin sulfate 40g/L, dextran 1 5g/L, neomycin 5mg/L, tune pH value to 7.2, osmotic pressure 400mOsm
2nd, intravascular stent is sealed in the polybag for filling protection liquid；
3rd, under the conditions of 500MPa high static pressures, handle 3 times, each time is 1 minute；
4th, after blood vessel is taken out, it is placed in the protection liquid containing 3% dodecyl sodium sulfate+2000U/ml DNA enzymatics, temperature
For 30 DEG C, shaking table speed is set as 100 revs/min, is handled 4 hours；
5th, after detergent and ferment treatment, take out Valved allograft and be placed in protection liquid and rinse 5 hours.
Claims (9)
1. a kind of new bioengineering takes off the preparation method of cell Valved allograft support, it is characterised in that comprises the following steps：
(1) animal artery blood vessel is taken；
(2) the hybrid protection liquid comprising one or more protection band valve vascular functions and tissue integrity is used；
(3) the endovascular cell of high static pressure pre-separation is used；
(4) smudge cells core is removed using composite nucleic acid enzyme；
(5) detergent removing loose crushing cell is utilized；
(6) rinsing removes unnecessary nuclease and cell fragment in Valved allograft hybrid protection liquid；
(7) the band valve blood of the de- cell with biological nature of three-dimensional structure and arrangement polarity with normal collagenous fibres is obtained
Pipe, wherein the hybrid protection liquid is into being grouped into：RPMI-1640 culture mediums；The concentration of chondroitin sulfate is 20-50g/L；It is right
The concentration for revolving sugared acid anhydride is 10-20g/L；Glycerine 100-200g/L；Neomycin 5-10mg/L；Regulation pH value is 6.8-7.2, osmotic pressure
For 300-400mOsm..
2. preparation method as claimed in claim 1, take aorta ascendens and active of the Valved allograft for fresh adult pig or ox
The coronary artery of arcus haemalis and 6-8mm length.
3. preparation method as claimed in claim 1, wherein the composite nucleic acid enzyme includes DNA enzymatic, RNase and other nucleases
And combinations thereof.
4. preparation method as claimed in claim 1, wherein the detergent includes dodecyl sodium sulfate, dodecyl sulphate
It is one or more as combination in sodium, sodium alginate, CHAPS activating agents.
5. preparation method as claimed in claim 1, wherein rinsing removes unnecessary nucleic acid in Valved allograft hybrid protection liquid
The time rinsed in enzyme and cell fragment is 2~5 hours.
6. preparation method as claimed in claim 1, the Valved allograft support of preparation need to be attached on nitroglycerin mould, be put into nothing
It is dehydrated in water calcium chloride molecular sieve, seals and mark through pack, the Gamma x ray irradiation xs sterilizing 5- for being 25kGy with irradiation dose
Saved backup after 10 hours at 4 DEG C.
7. preparation method as claimed in claim 1, wherein described static pressure pressure condition is 200-500MPa, high static pressure frequency
It it is every time 1~3 minute for 3-6 times.
8. preparation method as claimed in claim 3, wherein the composite nucleic acid enzyme is using the concentration of DNA enzymatic during DNA enzymatic
1000-2000U/ml, the use of concentration during nuclease is 1000-2000U/ml, the de- nucleus time of DNA enzymatic or nuclease is 2-
4 hours, treatment temperature was 15-30 degrees Celsius.
9. preparation method as claimed in claim 4, wherein the concentration of the detergent dodecyl sodium sulfate is 0.1-5%,
The concentration of lauryl sodium sulfate is 0.1-5%, and the concentration of sodium alginate is 0.1-5%, and detergent usage time is that 2-4 is small
When.
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| CN201610297151.7A CN105833356B (en) | 2016-05-09 | 2016-05-09 | A kind of new de- cell Valved allograft support and preparation method thereof |
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| CN105833356B true CN105833356B (en) | 2018-02-16 |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1762311A (en) * | 2005-09-08 | 2006-04-26 | 吴忠仕 | Preparation process of biological valve-possessed duct for pulmonary artery vessel restoration or reconstruction |
| 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 |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6734018B2 (en) * | 1999-06-07 | 2004-05-11 | Lifenet | Process for decellularizing soft-tissue engineered medical implants, and decellularized soft-tissue medical implants produced |
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2016
- 2016-05-09 CN CN201610297151.7A patent/CN105833356B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1762311A (en) * | 2005-09-08 | 2006-04-26 | 吴忠仕 | Preparation process of biological valve-possessed duct for pulmonary artery vessel restoration or reconstruction |
| 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 |
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Effective date of registration: 20211111 Address after: 611139 floors 1-4, building 15, No. 1919, Shuangyan Road, cross strait science and Technology Industrial Development Park, Wenjiang District, Chengdu, Sichuan Patentee after: Baiodisel (Chengdu) Biotechnology Co., Ltd Address before: 100176 601a, 6 / F, building 13, yard 8, Liangshuihe Second Street, Beijing Economic and Technological Development Zone, Beijing Patentee before: Baiodisel (Beijing) Biotechnology Co., Ltd |