CN109529120A - A kind of small intestinal submucosa matrix repairs the preparation method of gel - Google Patents
A kind of small intestinal submucosa matrix repairs the preparation method of gel Download PDFInfo
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- CN109529120A CN109529120A CN201811450324.XA CN201811450324A CN109529120A CN 109529120 A CN109529120 A CN 109529120A CN 201811450324 A CN201811450324 A CN 201811450324A CN 109529120 A CN109529120 A CN 109529120A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
- A61L27/3633—Extracellular matrix [ECM]
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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
- A61L27/14—Macromolecular materials
- A61L27/20—Polysaccharides
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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
- A61L27/3687—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
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
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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
- A61L27/52—Hydrogels or hydrocolloids
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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 present invention provides a kind of preparation method of small intestinal submucosa matrix reparation gel, comprising the following steps: carries out de- cell processing to small intestinal submucosa tissue, beats powder after freeze-drying；Small intestinal submucosa powder is dissolved in PBS buffer solution, then lysate is mixed with 1.5-3.5mol/L cellulose solution, 1.0-5.0mol/L epoxychloropropane solution, small intestinal submucosa matrix gel can be obtained after incubation at room temperature, the method for preparing gel is simple, mild condition, trophic factors and growth factor destruction in extracellular matrix is small, and repairing effect is good.
Description
Technical field
The present invention relates to technical field of biological materials, repair gel more particularly, to a kind of small intestinal submucosa matrix.
Background technique
The reparation of histoorgan and reconstruction refer to after tissue, organ damage repaired by closing on healthy cell by regenerating it is extensive
Multiple process, due to the defect for organizing spontaneous agglutination intrinsic, when defect or dysfunction occur in tissue and organ, just
It will cause aregeneratory either permanent defect to a certain extent.Therefore the reparation and reconstruction of histoorgan are always to give birth to
The focus of the related fieldss such as object, medicine.Along with the development to regeneration and restoration mechanism and organizational engineering research and deeply,
Find and develop it is various have the absorbable of application value and regenerated biological substance can be promoted, have become a heat in the field
Point.
Extracellular matrix (extracellular matrix, ECM) be by zooblast synthesis and secretion to it is extracellular, point
Macromolecular of the cloth between cell surface or cell, main component include collagenous fibres, glycoprotein, mucin etc., other compositions
There are the carbohydrates such as aminoglucan (hyaluronic acid, chondroitin sulfate), there are also some lipids and growth factors.These substances constitute multiple
Institutional framework is supported and connected to miscellaneous grid structure, adjusts the generation of tissue and the physiological activity of cell, in cell migration, divides
Change and proliferation aspect has important role.
Xenogenesis or variant cell antigen are due to being considered allochthon by host, having caused the inflammatory reaction of host and exempting from
The rejection that epidemic disease mediates.However extracellular matrix is the compound of structural proteins and functional protein, the component is usually in difference
It guards, and can be resistant to by heterologous receptor between species.Many animal tissue's organs and human body have similar extracellular matrix components
And structure, by take off cell technology obtain ECM biological support be widely used and tissue rebuild, as heart film valve,
The natural surroundings of skin, tendon and endocranium etc., three-dimensional structure and the growth of internal cell are close, can not only play bracket material
The effect of material, and include a variety of growth factors, there is important facilitation in tissue repair and in rebuilding.
The present invention is directed to the limitation of existing acellular matrix repair materials, and it is solidifying to provide a kind of small intestinal submucosa matrix
The preparation method of glue is handled by de- cell processing and gelation, eliminates the immunogenicity of xenogenesis (body) tissue, to greatest extent
The activity for remaining tissue extracelluar matrix ingredient, can specificity repair human body damaged tissues organ, inducing tissue regeneration,
And strong applicability is able to satisfy the needs of the various irregular shape restoring areas of different tissues.
Summary of the invention
The present invention provides a kind of preparation method of small intestinal submucosa matrix reparation gel, comprising the following steps:
S1. de- cell processing is carried out to small intestinal submucosa tissue, beats powder after freeze-drying；
S2. small intestinal submucosa powder is dissolved in PBS buffer solution, then by lysate and 1.5-3.5mol/L fiber
Plain solution, 1.0-5.0mol/L epoxychloropropane solution are mixed, and small intestinal submucosa matrix can be obtained after incubation at room temperature
Gel.
Preferably, the de- cell processing removes Cell membrane lipids ingredient, removing using the PBS buffer solution containing detergent
It is cleaned after processing using PBS buffer solution.
Preferably, the concentration of the detergent is 0.1~2wt%；The type of detergent is selected from triton x-100, deoxidation
Any one of sodium taurocholate, peregal and dodecyl sodium sulfate.
Preferably, it is to be freeze-dried small intestinal mucosa acellular matrix, then taken off with pulverizer handle that powder is beaten in the freeze-drying
Cellular matrix is clayed into power, and acellular matrix powder is obtained.
Preferably, lysate in mixed solution, cellulose solution obtained by S2, epoxychloropropane volume ratio be 1-5:1:1.
Preferably, the time of S2 incubation at room temperature is 24~48h.
Innovative point of the invention is:
(1) method for preparing gel is simple, mild condition, and the trophic factors and growth factor in extracellular matrix destroy
Small, repairing effect is good；
(2) small intestinal submucosa matrix and cellulose, epoxychloropropane are combined to be made for the first time and repair gel, it is maximum
The activity of the reservation extracellular matrix components of limit, highly-safe, strong applicability, repairing human body damaged tissues organ has specifically
Property, it being capable of inducing tissue regeneration reparation.
Obviously, above content according to the present invention is not being departed from according to the ordinary technical knowledge and customary means of this field
Under the premise of the above-mentioned basic fundamental thought of the present invention, the modification, replacement or change of other diversified forms can also be made.
The specific embodiment of form by the following examples remakes further specifically above content of the invention
It is bright.But the range that this should not be interpreted as to the above-mentioned theme of the present invention is only limitted to example below.It is all to be based on above content of the present invention
The technology realized all belongs to the scope of the present invention.
Specific embodiment
Embodiment 1
A kind of small intestinal submucosa matrix repairs the preparation method of gel, comprising the following steps:
The fresh chitterlings for butchering rear half an hour are taken, small intestine contents is washed away with water, overturns small intestine, after salt rubbing is added
With water repeated flushing 3 times, small intestine is splitted with scalpel, is then cut into the intestinal segment of 15 centimeter lengths.Muscle layer, serous coat are struck off with spatula
Layer, is placed in 4 DEG C of preservations in physiological saline and isolates SIS overnight.It is removed using the PBS buffer solution of the triton x-100 containing 1wt%
Cell membrane lipids ingredient is cleaned after removing processing using PBS buffer solution.Small intestinal mucosa acellular matrix is freeze-dried,
Acellular matrix is clayed into power with pulverizer again, obtains small intestinal submucosa matrix powder.By small intestinal submucosa matrix powder
End, which is dissolved in PBS buffer solution, obtains lysate, then by lysate and 1.5mol/L cellulose solution, 1.0mol/L epoxy chloropropionate
Alkane solution is mixed, lysate in gained mixed solution, cellulose solution, epoxychloropropane volume ratio be 1:1:1, room temperature
Small intestinal submucosa matrix gel can be obtained after incubation, the time of incubation is for 24 hours.
It will be implanted into male SD rat dorsal sc after gel ethylene oxide sterilizing will be repaired, in 1,2,4,8 week point after implantation
Not Qu Cai (n=3) carry out histological observation, carry out inflammatory cell counting.The experimental results showed that repair materials group inflammatory of the present invention
Cell number maintains reduced levels, is basically unchanged, and has good histocompatbility, can be used for repairing in vivo.
Embodiment 2
A kind of small intestinal submucosa matrix repairs the preparation method of gel, comprising the following steps:
The fresh chitterlings for butchering rear half an hour are taken, small intestine contents is washed away with water, overturns small intestine, after salt rubbing is added
With water repeated flushing 3 times, small intestine is splitted with scalpel, is then cut into the intestinal segment of 15 centimeter lengths.Muscle layer, serous coat are struck off with spatula
Layer, is placed in 4 DEG C of preservations in physiological saline and isolates SIS overnight.It is thin using the PBS buffer solution removing of the peregal containing 0.1wt%
After birth lipid components are cleaned after removing processing using PBS buffer solution.Small intestinal mucosa acellular matrix is freeze-dried, then
Acellular matrix is clayed into power with pulverizer, obtains small intestinal submucosa matrix powder.By small intestinal submucosa matrix powder
It is dissolved in PBS buffer solution and obtains lysate, then by lysate and 1.5mol/L cellulose solution, 5.0mol/L epoxychloropropane
Solution is mixed, lysate in gained mixed solution, cellulose solution, epoxychloropropane volume ratio be 3:1:1, room temperature is incubated
Small intestinal submucosa matrix gel can be obtained after educating, the time of incubation is 36h.
It will be implanted into male SD rat dorsal sc after gel ethylene oxide sterilizing will be repaired, in 1,2,4,8 week point after implantation
Not Qu Cai (n=3) carry out histological observation, carry out inflammatory cell counting.The experimental results showed that repair materials group inflammatory of the present invention
Cell number maintains reduced levels, is basically unchanged, and has good histocompatbility, can be used for repairing in vivo.
Embodiment 3
A kind of small intestinal submucosa matrix repairs the preparation method of gel, comprising the following steps:
The fresh chitterlings for butchering rear half an hour are taken, small intestine contents is washed away with water, overturns small intestine, after salt rubbing is added
With water repeated flushing 3 times, small intestine is splitted with scalpel, is then cut into the intestinal segment of 15 centimeter lengths.Muscle layer, serous coat are struck off with spatula
Layer, is placed in 4 DEG C of preservations in physiological saline and isolates SIS overnight.Using the PBS buffer solution of the dodecyl sodium sulfate containing 1wt%
Cell membrane lipids ingredient is removed, is cleaned after removing processing using PBS buffer solution.It is dry that small intestinal mucosa acellular matrix is subjected to freezing
It is dry, then acellular matrix is clayed into power with pulverizer, obtain small intestinal submucosa matrix powder.By small intestinal submucosa matrix
Powder, which is dissolved in PBS buffer solution, obtains lysate, then by lysate and 3.5mol/L cellulose solution, 1.0mol/L epoxy chlorine
Propane solution is mixed, lysate in gained mixed solution, cellulose solution, epoxychloropropane volume ratio be 4:1:1, room
Small intestinal submucosa matrix gel can be obtained in temperature after being incubated for, the time of incubation is 36h.
It will be implanted into male SD rat dorsal sc after gel ethylene oxide sterilizing will be repaired, in 1,2,4,8 week point after implantation
Not Qu Cai (n=3) carry out histological observation, carry out inflammatory cell counting.The experimental results showed that repair materials group inflammatory of the present invention
Cell number maintains reduced levels, is basically unchanged, and has good histocompatbility, can be used for repairing in vivo.
Embodiment 4
A kind of small intestinal submucosa matrix repairs the preparation method of gel, comprising the following steps:
The fresh chitterlings for butchering rear half an hour are taken, small intestine contents is washed away with water, overturns small intestine, after salt rubbing is added
With water repeated flushing 3 times, small intestine is splitted with scalpel, is then cut into the intestinal segment of 15 centimeter lengths.Muscle layer, serous coat are struck off with spatula
Layer, is placed in 4 DEG C of preservations in physiological saline and isolates SIS overnight.It is removed using the PBS buffer solution of the NaTDC containing 2wt%
Cell membrane lipids ingredient is cleaned after removing processing using PBS buffer solution.Small intestinal mucosa acellular matrix is freeze-dried,
Acellular matrix is clayed into power with pulverizer again, obtains small intestinal submucosa matrix powder.By small intestinal submucosa matrix powder
End, which is dissolved in PBS buffer solution, obtains lysate, then by lysate and 2.0mol/L cellulose solution, 4.0mol/L epoxy chloropropionate
Alkane solution is mixed, lysate in gained mixed solution, cellulose solution, epoxychloropropane volume ratio be 5:1:1, room temperature
Small intestinal submucosa matrix gel can be obtained after incubation, the time of incubation is 48h.
It will be implanted into male SD rat dorsal sc after gel ethylene oxide sterilizing will be repaired, in 1,2,4,8 week point after implantation
Not Qu Cai (n=3) carry out histological observation, carry out inflammatory cell counting.The experimental results showed that repair materials group inflammatory of the present invention
Cell number maintains reduced levels, is basically unchanged, and has good histocompatbility, can be used for repairing in vivo.
It is demonstrated experimentally that small intestinal submucosa matrix gel of the present invention, can be effective against the degradation of acid and pepsin, power
Function admirable is learned, no cytotoxicity, hemolysis rate is low, and histocompatbility is good, and pore size is suitable, is suitable for cell and grows, simultaneously
It containing the raised growth factor, repairs in vivo without obvious calcification phenomenon, can be used for promoting gastrointestinal mucosal reparation, use is safe, system
Preparation Method is simple, has good clinical value.
Claims (6)
1. the preparation method that a kind of small intestinal submucosa matrix repairs gel, which comprises the following steps:
S1. de- cell processing is carried out to small intestinal submucosa tissue, beats powder after freeze-drying；
S2. small intestinal submucosa powder is dissolved in PBS buffer solution, it is then that lysate and 1.5-3.5mol/L cellulose is molten
Liquid, 1.0-5.0mol/L epoxychloropropane solution are mixed, and small intestinal submucosa matrix gel can be obtained after incubation at room temperature.
2. the preparation method that a kind of small intestinal submucosa matrix according to claim 1 repairs gel, which is characterized in that institute
De- cell processing is stated using the PBS buffer solution removing Cell membrane lipids ingredient containing detergent, is buffered after removing processing using PBS
Liquid cleaning.
3. the preparation method that a kind of small intestinal submucosa matrix according to claim 2 repairs gel, which is characterized in that institute
The concentration for stating detergent is 0.1~2wt%；The type of detergent is selected from triton x-100, NaTDC, peregal, and
Any one of dodecyl sodium sulfate.
4. the preparation method that a kind of small intestinal submucosa matrix according to claim 2 repairs gel, which is characterized in that institute
Stating freeze-drying and beating powder is to be freeze-dried small intestinal mucosa acellular matrix, then acellular matrix is clayed into power with pulverizer,
Obtain acellular matrix powder.
5. the preparation method that a kind of small intestinal submucosa matrix according to claim 1 repairs gel, which is characterized in that S2
Lysate in gained mixed solution, cellulose solution, epoxychloropropane volume ratio be 1-5:1:1.
6. the preparation method that a kind of small intestinal submucosa matrix according to claim 1 repairs gel, which is characterized in that S2
The time of incubation at room temperature is 24~48h.
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| CN201811450324.XA CN109529120A (en) | 2018-11-30 | 2018-11-30 | A kind of small intestinal submucosa matrix repairs the preparation method of gel |
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| CN201811450324.XA CN109529120A (en) | 2018-11-30 | 2018-11-30 | A kind of small intestinal submucosa matrix repairs the preparation method of gel |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114540267A (en) * | 2022-01-27 | 2022-05-27 | 广东省农业科学院动物科学研究所 | In-vitro preparation method of collagen networks with different concentrations for intestinal mucosa of piglets |
| CN116492508A (en) * | 2023-02-28 | 2023-07-28 | 诺一迈尔（苏州）医学科技有限公司 | Injectable hydrogel for promoting articular cartilage repair and preparation method thereof |
| CN117797172A (en) * | 2024-02-28 | 2024-04-02 | 合肥启色生物科技有限公司 | Preparation method of anti-inflammatory, hemostatic and healing-promoting combination |
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| US20110117167A1 (en) * | 2009-11-18 | 2011-05-19 | Affinergy, Inc. | Methods and compositions for soft tissue repair |
| CN103599567A (en) * | 2013-11-25 | 2014-02-26 | 四川大学华西医院 | Thermo-sensitive composite material and preparation method and application thereof |
| CN104971380A (en) * | 2014-04-11 | 2015-10-14 | 烟台隽秀生物科技有限公司 | Acellular matrix repairing gel and new method for preparing the same |
| CN105056306A (en) * | 2013-04-17 | 2015-11-18 | 四川大学华西医院 | Application of material prepared by genipin cross-linking intestinal submucosa |
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| US20110117167A1 (en) * | 2009-11-18 | 2011-05-19 | Affinergy, Inc. | Methods and compositions for soft tissue repair |
| CN105056306A (en) * | 2013-04-17 | 2015-11-18 | 四川大学华西医院 | Application of material prepared by genipin cross-linking intestinal submucosa |
| CN103599567A (en) * | 2013-11-25 | 2014-02-26 | 四川大学华西医院 | Thermo-sensitive composite material and preparation method and application thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114540267A (en) * | 2022-01-27 | 2022-05-27 | 广东省农业科学院动物科学研究所 | In-vitro preparation method of collagen networks with different concentrations for intestinal mucosa of piglets |
| CN116492508A (en) * | 2023-02-28 | 2023-07-28 | 诺一迈尔（苏州）医学科技有限公司 | Injectable hydrogel for promoting articular cartilage repair and preparation method thereof |
| CN117797172A (en) * | 2024-02-28 | 2024-04-02 | 合肥启色生物科技有限公司 | Preparation method of anti-inflammatory, hemostatic and healing-promoting combination |
| CN117797172B (en) * | 2024-02-28 | 2024-05-14 | 合肥启色生物科技有限公司 | Preparation method of anti-inflammatory, hemostatic and healing-promoting combination |
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