CN106496620A - A kind of modified collagen and preparation method thereof - Google Patents
A kind of modified collagen and preparation method thereof Download PDFInfo
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- CN106496620A CN106496620A CN201611025503.XA CN201611025503A CN106496620A CN 106496620 A CN106496620 A CN 106496620A CN 201611025503 A CN201611025503 A CN 201611025503A CN 106496620 A CN106496620 A CN 106496620A
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- collagen
- modified collagen
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- tea polyphenols
- collagem
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J7/00—Chemical treatment or coating of shaped articles made of macromolecular substances
-
- 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
- A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
- A61L27/24—Collagen
-
- 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
- A61L27/54—Biologically active materials, e.g. therapeutic substances
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J3/00—Processes of treating or compounding macromolecular substances
- C08J3/24—Crosslinking, e.g. vulcanising, of macromolecules
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J5/00—Manufacture of articles or shaped materials containing macromolecular substances
- C08J5/18—Manufacture of films or sheets
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J7/00—Chemical treatment or coating of shaped articles made of macromolecular substances
- C08J7/12—Chemical modification
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
- C08L89/00—Compositions of proteins; Compositions of derivatives 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
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
- A61L2300/216—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials with other specific functional groups, e.g. aldehydes, ketones, phenols, quaternary phosphonium groups
-
- 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
- A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J2389/00—Characterised by the use of proteins; Derivatives thereof
Abstract
The invention provides a kind of modified collagen, preparation method is as follows：With culture plate as mould, collagen protein 2ml is taken, it is collagem membrane to be prepared in 10%w/v acetums to add concentration；According to 1000mg：The 300mg collagem membranes of preparation are placed in precrosslink in 0.64%w/v tea polyphenols solution by the ratio of 1ml, are soaked in the 0.05 mol/L acetums containing 0.64%w/v tea polyphenols, crosslinking, and cleaning is obtained final product after drying.It is an advantage of the current invention that：Prepared modified collagen has the aggregation that more preferable ability induces repairing type macrophage, improve the biological property of collagem membrane, the bio-toxicity of medicine collagen composite membrane is reduced, and with good application prospect, new selection is provided for the related increment operation such as guiding regeneration art.
Description
Technical field
The invention belongs to technical field of biological material, and in particular to a kind of modified collagen and preparation method thereof.
Background technology
Often need to apply Guided Bone Regeneration operation to carry out bone increment in dental operation.Collagen be extracellular matrix one kind into
Point, have been widely used in guided bone regeneration.Collagem membrane has preferable bio-compatibility, and osteoblast can be promoted to stick
Attached, propagation, migration and break up, can also have stop fibroblast quickly invade bone growth site, separate soft tissue with hard
The barrier functions such as the growth of tissue.
However, used as a kind of exogenous graft thing, collagen still can cause certain inflammatory reaction, at the same also because it
Readily degradable and lack preferable mechanical performance.Commonly used in the art be crosslinked with collagen using glutaraldehyde, so as to improve its
Mechanical performance, but easily cause inflammation and produce cytotoxicity.Therefore, the long-term preservation of collagem membrane and use, necessary not only for
Mechanical performance is improved, antiinflammatory is more wanted and is improved bio-compatibility.
Tea polyphenols（Catchine）It is a kind of polyphenol from green tea, it has numerous biological functions, extensively receives in recent years
Concern.Scientists find that tea polyphenols have anticancer, antioxidation, anti-inflammatory, fibrosis and promote the effect of skeletonization.Meanwhile,
As a kind of modifying agent of collagen, Chu et al., it can also report which can both strengthen the mechanical performance of collagen, will not also destroy
The triple helix structure of collagen.Therefore, EGCG- collagen composite membranes can be used in the operation such as Guided Bone Regeneration, and have good
Prospect.
The report such as Sadtler, effect of the collagen with induction repairing type macrophage, can promote M2 in the recent period（Repairing type
Macrophage）The aggregation of cell and the repairing type path of mTOR mediations, and then promote the reparation of muscular tissue.
Tea polyphenols incorporating collagen film is wanted to alleviate due to inflammatory reaction caused by foreign body by the present invention, while inducing repairing type
The aggregation of macrophage, and then promotion organization reparation.
Content of the invention
The purpose of the present invention is for the deficiencies in the prior art, there is provided a kind of modified collagen and preparation method thereof.
In order to achieve the above object, following technical proposal present invention employs：A kind of modified collagen, preparation method are as follows：
A, with culture plate as mould, take collagen protein 2ml, it is to prepare collagem membrane in 10%w/v acetums to add concentration；
B, according to 1000mg：The 300mg collagem membranes of preparation are placed in precrosslink in 0.64%w/v tea polyphenols solution by the ratio of 1ml,
The 0.05 mol/L acetums containing 0.64%w/v tea polyphenols are soaked in, are crosslinked, cleaning is obtained final product after drying.
Preferably, the collagen protein is Niu Laiyuan type i collagens.
Preferably, in step A, freeze overnight at a temperature of -20 DEG C, under the conditions of -53 DEG C, 1 Pa, vacuum is cold
The dry 24h of lyophilizing.
Preferably, in step B, the precrosslink time is 10-60min.
Preferably, in step B, crosslinking time is 24h.
Preferably, in step B, by the tea polyphenols-collagen composite membrane lyophilization under the conditions of -20 DEG C for preparing
Overnight.
It is an advantage of the current invention that：It is thin that modified collagen prepared by the present invention has more preferable ability induction repairing type macrophage
The aggregation of born of the same parents, improves the biological property of collagem membrane, reduces the bio-toxicity of medicine-collagen composite membrane, answers with good
With prospect, new selection is provided for the related increment operation such as guiding regeneration art.
Description of the drawings
Fig. 1 is 3 flow cytometry tests result figure of embodiment.
Fig. 2 is 3 polymerase chain reaction experimental result picture of embodiment.
Fig. 3 is 3 polymerase chain reaction experimental result picture of embodiment.
Fig. 4 is embodiment 3HE Coloration experiment result figure.
Fig. 5 is embodiment 3Masson Coloration experiment result figure.
Specific embodiment
Below in conjunction with the accompanying drawings, the present invention is described in detail.
In order that the objects, technical solutions and advantages of the present invention become more apparent, below in conjunction with drawings and Examples, right
The present invention is further elaborated.It should be appreciated that specific embodiment described herein is only in order to explain the present invention, and
It is not used in the restriction present invention.
Embodiment 1：As mould, it is 10% in concentration to add collagen protein 2mL to culture plate with a diameter of 3.5cm/ holes（w/
v）Acetum.- 26 DEG C of cryogenic refrigerator freeze overnights, under the conditions of -53 DEG C, 1Pa, prepared by freeze dryer vacuum lyophilization 24h
Collagem membrane.
2. tea polyphenols（0.64%）Gradient concentration solution is prepared.
3. the modified collagem membrane of tea polyphenols
The collagem membrane of preparation is placed in 0.64% tea polyphenols after 10 min of precrosslink, 0.05 containing 0.64% tea polyphenols is soaked in
Mol/L acetums, are crosslinked 24 h in 4 DEG C, and l X PBSs are rinsed 3 times.
By the collagem membrane for preparing under the conditions of -20 DEG C lyophilized overnight.
Embodiment 2：A kind of modified collagen, preparation method are as follows：
A, with culture plate as mould, take collagen protein 2ml, it is to prepare collagem membrane in 10%w/v acetums to add concentration；
B, according to 1000mg：The 300mg collagem membranes of preparation are placed in precrosslink in 0.64%w/v tea polyphenols solution by the ratio of 1ml,
The 0.05 mol/L acetums containing 0.64%w/v tea polyphenols are soaked in, are crosslinked, cleaning is obtained final product after drying.
The collagen protein is Niu Laiyuan type i collagens.
In step A, freeze overnight at a temperature of -20 DEG C, under the conditions of -53 DEG C, 1 Pa, vacuum lyophilization 24h.
In step B, the precrosslink time is 10-60min.
In step B, crosslinking time is 24h.
In step B, by the tea polyphenols-collagen composite membrane for preparing under the conditions of -20 DEG C lyophilized overnight.
Embodiment 3：Bio-compatibility is tested
1. flow cytometry
Male SD rat by 6-8 week old（Weight 250-300g）27 are randomly divided into 9 groups, with 10% chloral hydrate lumbar injection fiber crops
After liquor-saturated, in the otch that every rat back makes 3 long 1mm, corresponding material is implanted into（1mm, circular）Afterwards with the nonabsorable of 3-0
Nylon suture sewing-up cut.Mouse is put to death respectively with dislocation of cervical vertebra method the 3rd day, the 5th day, the 7th day, is straight with 1mm otch
Footpath ring-type removes full thickness skin tissue, is rinsed with PBS immediately, adds type i collagen enzyme, II Collagenase Types after shredding（0.25mg/
ml）Digestion tissue in the environment of 36.5 degrees Celsius, filters tissue suspension with cell screen clothes after 2 hours, at 1300 turns per minute
The lower centrifugation of speed 3 minutes, then rinsed with PBS, add CD206 antibody immediately on ice, after lucifuge incubation half an hour, per minute
It is centrifuged 3 minutes under 1300 rotating speeds, add PBS and FITC labellings after removing suspension two resist, after incubation half an hour on ice,
It is centrifuged 3 minutes under 1300 rotating speed per minute, after removing suspension, adds PBS, going up machine immediately carries out Flow cytometry.
As shown in figure 1, Flow cytometry material is implanted into the subcutaneous rat CD206 positive（Repairing type macrophage mark
Thing）Ratio, as a result point out：Compared to matched group and the group of collagem membrane is applied, the modified collagem membrane of tea polyphenols is significantly lured
More repairing type macrophages are led.
2. real-time quantitative polymerase chain reaction (qPCR)
As shown in Fig. 2 material is implanted into rat calvarium Cranial defect（Circle, diameter：5mm）It has been obviously improved CD163, CD206（Repair
Type macrophage marker）Expression, and bmp2, runx2, opn, col1a1（Bone formation-related gene）.
As shown in figure 3, material is implanted into rat calvarium Cranial defect（Circle, diameter：5mm）, relative to collagem membrane group（Col）,
The modified collagem membrane of tea polyphenols（E-Col）The expression of the growth factor gene for repairing related is obviously promoted, including Ink vessel transfusing
Skin growth factor（VEGF）, basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF-A, PDGF-B),
Insulin like growth factor（IGF-1）, transforming growth factor-β（TGF-β）Expression.
3. HE dyeing
As shown in figure 4, the modified collagem membrane group of tea polyphenols is implanted into rat calvarium Cranial defect（Circle, diameter：5mm）, hence it is evident that promote
Enter osteanagenesis.
4. Masson dyeing
As shown in figure 5, the modified collagem membrane group of tea polyphenols is obviously promoted the deposition of collagen.
The invention is not limited in aforesaid specific embodiment.The present invention is expanded to and any is disclosed in this manual
New feature or any new combination, and the arbitrary new method that discloses or the step of process or any new combination.
Claims (7)
1. a kind of modified collagen, it is characterised in that preparation method is as follows：
A, with culture plate as mould, take collagen protein 2ml, it is to prepare collagem membrane in 10%w/v acetums to add concentration；
B, according to 1000mg：The 300mg collagem membranes of preparation are placed in precrosslink in 0.64%w/v tea polyphenols solution by the ratio of 1ml,
The 0.05 mol/L acetums containing 0.64%w/v tea polyphenols are soaked in, are crosslinked, cleaning is obtained final product after drying.
2. a kind of modified collagen according to claim 1, it is characterised in that：The collagen protein is Niu Laiyuan type i collagens.
3. a kind of modified collagen according to claim 1, it is characterised in that：In step A, cold at a temperature of -26 DEG C
Freeze overnight, under the conditions of -53 DEG C, 1Pa, vacuum lyophilization 24h.
4. a kind of modified collagen according to claim 1, it is characterised in that：In step B, the precrosslink time is 10-
60min.
5. a kind of modified collagen according to claim 1, it is characterised in that：In step B, crosslinking time is 24h.
6. a kind of modified collagen according to claim 1, it is characterised in that：In step B, will be many for the tea for preparing
Phenol-collagen composite membrane lyophilized overnight under the conditions of -20 DEG C.
7. according to a kind of arbitrary described modified collagen of claim 1-6, it is characterised in that：The modified collagen is in medical field
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| CN201611025503.XA CN106496620A (en) | 2016-11-22 | 2016-11-22 | A kind of modified collagen and preparation method thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108744037A (en) * | 2018-06-29 | 2018-11-06 | 四川大学 | A kind of modified interleukin collagem membrane and its application |
| CN108853586A (en) * | 2018-06-29 | 2018-11-23 | 四川大学 | A kind of modified tea polyphenol collagem membrane and its application |
| CN114259602A (en) * | 2021-12-29 | 2022-04-01 | 江苏江山聚源生物技术有限公司 | Recombinant collagen hydrogel for injection and preparation method thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105288734A (en) * | 2015-11-23 | 2016-02-03 | 四川大学 | Composite crosslinked type I collagen membrane and preparation method thereof |
-
2016
- 2016-11-22 CN CN201611025503.XA patent/CN106496620A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105288734A (en) * | 2015-11-23 | 2016-02-03 | 四川大学 | Composite crosslinked type I collagen membrane and preparation method thereof |
Non-Patent Citations (1)
| Title |
|---|
| 党美珠: "天然多酚改性鱼鳞胶原蛋白-壳聚糖膜复合材料的制备及其应用研究", 《中国优秀硕士学位论文全文数据库•医药卫生科技辑》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108744037A (en) * | 2018-06-29 | 2018-11-06 | 四川大学 | A kind of modified interleukin collagem membrane and its application |
| CN108853586A (en) * | 2018-06-29 | 2018-11-23 | 四川大学 | A kind of modified tea polyphenol collagem membrane and its application |
| CN114259602A (en) * | 2021-12-29 | 2022-04-01 | 江苏江山聚源生物技术有限公司 | Recombinant collagen hydrogel for injection and preparation method thereof |
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