CN101810881B - Preparation method of sericin/hydroxyapatite composite porous bracket - Google Patents
Preparation method of sericin/hydroxyapatite composite porous bracket Download PDFInfo
- Publication number
- CN101810881B CN101810881B CN 201010157889 CN201010157889A CN101810881B CN 101810881 B CN101810881 B CN 101810881B CN 201010157889 CN201010157889 CN 201010157889 CN 201010157889 A CN201010157889 A CN 201010157889A CN 101810881 B CN101810881 B CN 101810881B
- Authority
- CN
- China
- Prior art keywords
- sericin
- solution
- preparation
- hydroxyapatite
- composite porous
- 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.)
- Expired - Fee Related
Links
Images
Abstract
The invention discloses a preparation method of a sericin/hydroxyapatite composite porous bracket. The method comprises the following steps: respectively synthesizing hydroxyapatite powder and preparing a sericin solution, mixing according to different mass proportions, and using the freeze-drying method to prepare a sericin/hydroxyapatite composite porous bracket material. The prepared sericin/hydroxyapatite composite porous bracket material has the advantages of simple preparation process, short preparation time, easily-satisfied preparation conditions, no porogenic agent addition, less and cheap reagent and low cost. Besides, the prepared bracket material has higher porosity and mechanical property, and the pores are uniformly dispersed and are communicated mutually.
Description
Technical field
The present invention relates to the processing and utilization field of sericin, particularly sericin/hydroxyapatite composite porous bracket material preparation method in a kind of bone tissue engineer.
Background technology
Bone injury and bone are damaged to be disease very common in the daily life.Traditional medicine mainly adopts from the method for body and heteroplastic transplantation carries out the damaged reparation of bone, but this method occurs the drawbacks such as new damage and the reflection of generation immunologic rejection, is not rational bone restorative procedure.And bone tissue engineer is with a small amount of histiocyte repairing bone defect behind cultured and amplified in vitro, can reach perfect form reparation according to any plastotype of the damaged situation of bone simultaneously, has opened up new approach for realizing the not damaged reparation.
Sericin (Silk Sericin is called for short SS) is a kind of globular preteins, and relative molecular weight is 1.4～31.4 ten thousand, is comprised of 18 seed amino acids.Sericin has good water solublity, water absorption, biocompatibility, and the activity of energy restraint of tyrosinase has the characteristics such as non-oxidizability, anticoagulation, antitumor.As a kind of native protein resource, sericin can be applicable to biomaterial and field of medicaments etc.
Hydroxyapatite (hydroxyapatite, HA, Fig. 1) is a kind of bioactive ceramics, and its chemical composition and crystalline texture are similar to the apatite in the skeleton.Exchange reaction can occur with the aminoacid that contains carboxyl, protein, organic acid etc. in the Ca2+ in the HA molecule, have good bone conduction performance and biological activity, forming firmly with osseous tissue, synostosis promotes bone growth, phase is more stable, avirulence, inflammatory are the well behaved implantations of generally acknowledging.Fragility is large, toughness is relatively poor, mechanical property is not enough but also exist, and degraded slowly and easily from shortcomings such as implantation site migrations, this has all limited its use as human material implantation body.
Summary of the invention
The objective of the invention is respectively synthesizing hydroxylapatite powder and preparation sericin solution, both are mixed in the different quality ratio, prepare the sericin/hydroxyapatite composite porous bracket material by freeze-drying method.
The preparation method of a kind of sericin/hydroxyapatite composite porous bracket of the present invention may further comprise the steps:
(1) measures a certain amount of sericin solution and place beaker, in proportion to wherein adding hydroxyapatite powder, keep sericin consistent with the ratio of total amount in solution of hydroxyapatite;
(2) mixed solution of sericin and hydroxyapatite is put into ultrasonic cleaner, oscillation treatment 3～5min;
(3) taking out mixed solution, is 0.5% glutaraldehyde cross-linking agent to the concentration that wherein slowly drips trace, and magnetic stirring apparatus slowly stirs;
(4) mixed solution is moved into 96 porocyte culture plates fast, and put into immediately-20 ℃ of refrigerator precooling 12h;
(5) freezing 12h rear plate sample is taken out, under 20 ℃ of conditions of room temperature, rise again and process 4h;
(6) flat samples after will rising again is again put into-20 ℃ of refrigerator and cooled and is frozen, and then puts into-80 ℃ of freezer dryer lyophilizations, makes the sericin/hydroxyapatite composite porous bracket of different proportion.
Described sericin solution, its preparation method may further comprise the steps:
(1) will remove Bombyxmori Linnaeus cocoon layer behind the pupa, and put into boiling water and carry out degumming process;
(2) behind the 30min, stop steaming and decocting, filter roguing;
(3) to put into Rotary Evaporators concentrated for the sericin solution after will filtering, and obtains the sericin solution of purpose concentration.
Described hydroxylapatite powder, its preparation method may further comprise the steps:
(1) gets saturated Ca (OH)
2Solution places 60 ℃ of water-baths, drips the H of 10%wt
3PO
4Solution, and Ca (OH)
2Solution and H
3PO
4The volume ratio of solution is 70: 1～2, during continuous rapid stirring and keep water temperature;
(2) to drip the method for ammonia, the pH that adjusts solution remains on 8.7;
(3) drip H
3PO
4Continue to stir 3h behind the solution, stirring finishes to be placed on room temperature and leaves standstill 48h;
(4) the bottom precipitation is collected in the supernatant of inclining; Precipitation is cleaned 2-3 time with dehydrated alcohol first, cleans 3-4 time with distilled water again;
The precipitation that (5) will obtain is put into refrigerator and cooled and is frozen, and then moves in the freeze dryer dryly, grinds, and obtains hydroxyapatite powder.
The weight ratio of sericin and hydroxyapatite is 10: 1～1: 1 in a kind of preparation method of sericin/hydroxyapatite composite porous bracket, described step (1).
The preparation method of sericin solution, the consumption of water is to the cocoon layer submergence in the described step (1).
The preparation method of sericin solution, also comprise the sericin solution that obtains purpose concentration after, extract sericin solution, carry out sds gel electrophoresis, measure the sericin molecular weight.
The preparation method of hydroxylapatite powder, wherein step (1) is to get the saturated Ca of 700ml (OH)
2Solution places 60 ℃ of water-baths, drips the H of 10%wt
3PO
4Solution 11ml, during use the continuous rapid stirring of magnetic stirring apparatus and keep water temperature.
Usefulness of the present invention is:
Sericin and hydroxyapatite are made the bone tissue engineer compound support frame material, and sericin can be used as the binding agent of hydroxyapatite particle on the one hand, suppresses the hydroxyapatite particle in disengaging and the migration of implanting point; The mechanical property of material be can improve on the other hand, the toughness of polymer and intensity and the hardness of inorganic particulate obtained.
The present invention adopts blending method to prepare the sericin/hydroxyapatite composite porous bracket material, and comprehensive both premium properties obtain a kind of biological activity bone renovating material of function admirable.
The molecular weight ranges of the sericin that the present invention is used is 97.2～200.0KDa, and the sericin/hydroxyapatite composite porous bracket material preparation process of preparation is simple, takes weak point, and preparation condition satisfies easily; Do not add porogen, agents useful for same is few and cheap, and cost is low; The prepared timbering material porosity of the present invention is higher, hole be uniformly dispersed and Kong Yukong between mutually connect; Has certain mechanical property.
Description of drawings
Fig. 1 is the pore structure photo of hydroxyapatite.
Fig. 2 is that the concentration ratio of inventive embodiments is the pore structure photo of 9: 1 sericin/hydroxyapatite composite porous bracket material.
Fig. 3 is that the concentration ratio of inventive embodiments is the pore structure photo of 7: 3 sericin/hydroxyapatite composite porous bracket material.
Fig. 4 is that the concentration ratio of inventive embodiments is the pore structure photo of 5: 5 sericin/hydroxyapatite composite porous bracket material.
The specific embodiment
Below in conjunction with embodiment technical scheme of the present invention is described:
Embodiment 1: the preparation method of sericin solution may further comprise the steps:
(1) will remove Bombyxmori Linnaeus cocoon layer behind the pupa, and put into boiling water and carry out degumming process; The consumption of water is being advisable the cocoon layer submergence;
(2) behind the 30min, stop steaming and decocting, filter roguing;
(3) to put into Rotary Evaporators concentrated for the sericin solution after will filtering, and obtains the sericin solution of purpose concentration;
(4) extract sericin solution, carry out sds gel electrophoresis, measure the sericin molecular weight, the molecular weight ranges of sericin is 97.2～200.0KDa.
The preparation method of embodiment 2, hydroxylapatite powder may further comprise the steps:
(1) gets the saturated Ca of 700ml (OH)
2Solution places 60 ℃ of water-baths, drips the H of 10%wt
3PO
4Solution 11ml, during use the continuous rapid stirring of magnetic stirring apparatus and keep water temperature;
(2) adopt the digital display pH meter to monitor the pH of solution, to drip the method for ammonia, the pH that adjusts solution remains on 8.7;
(3) drip H
3PO
4Continue behind the solution with faster rotating speed stirring 3h, stirring finishes to be placed on room temperature and leaves standstill 48h;
(4) the bottom precipitation is collected in the supernatant of inclining; Precipitation is cleaned 2-3 time with dehydrated alcohol first, cleans 3-4 time with distilled water again;
The precipitation that (5) will obtain is put into refrigerator and cooled and is frozen, and then moves in the freeze dryer dryly, grinds, and obtains hydroxyapatite powder.
The preparation method of embodiment 3, a kind of sericin/hydroxyapatite composite porous bracket may further comprise the steps:
(1) measures a certain amount of sericin solution and place beaker, in proportion to wherein adding hydroxyapatite powder, keep sericin consistent with the ratio of total amount in solution of hydroxyapatite;
(2) mixed solution of sericin and hydroxyapatite is put into ultrasonic cleaner, oscillation treatment 3～5min;
(3) taking out mixed solution, is 0.5% glutaraldehyde cross-linking agent to the concentration that wherein slowly drips trace, and magnetic stirring apparatus slowly stirs;
(4) mixed solution is moved into 96 porocyte culture plates fast, and put into immediately-20 ℃ of refrigerator precooling 12h;
(5) freezing 12h rear plate sample is taken out, under 20 ℃ of conditions of room temperature, rise again and process 4h;
(6) flat samples after will rising again is again put into-20 ℃ of refrigerator and cooled and is frozen, and then puts into-80 ℃ of freezer dryer lyophilizations, makes the sericin/hydroxyapatite composite porous bracket of different proportion.
The Mechanics Performance Testing of embodiment 4, sericin/hydroxyapatite composite material
Adopt universal testing machine (AGS-J type, SHIMADZU company, Japan) each sample to be carried out the mensuration of elastic modelling quantity.Diameter 5mm is made in each timbering material unification, and high 5mm's is cylindric, the support upper and lower surface is parallel to each other and as far as possible smooth.Ambient temperature and humidity are consistent.Every group of sample got three Duplicate Samples, averages.
The preparation method of embodiment 5, a kind of sericin/hydroxyapatite composite porous bracket may further comprise the steps:
(1) a certain amount of sericin solution of accurate measuring places beaker, in proportion to wherein adding hydroxyapatite powder.Make HA: the ratio of SS is 1: 9.
(2) mixed solution of sericin and hydroxyapatite is put into ultrasonic cleaner, oscillation treatment 3～5min.
(3) taking out mixed solution, is 0.5% glutaraldehyde cross-linking agent to the concentration that wherein slowly drips trace, and magnetic stirring apparatus slowly stirs.
(4) mixed solution is moved into 96 porocyte culture plates fast, and put into immediately-20 ℃ of refrigerator precooling 12h.
(5) freezing 12h rear plate is taken out, at ambient temperature, rise again and process 4h, the sample of molding is taken out carefully.
(6) sample is put into again-20 ℃ of refrigerator and cooled and frozen, then put into-80 ℃ of freezer dryer lyophilizations, finally make sericin/hydroxyapatite composite porous bracket HA10.Its density is 33mg/ml, and porosity is 50.1%, and elastic modelling quantity is 7.41MPa, Fig. 2.
The preparation method of embodiment 6, a kind of sericin/hydroxyapatite composite porous bracket may further comprise the steps:
(1) a certain amount of sericin solution of accurate measuring places beaker, in proportion to wherein adding hydroxyapatite powder.Make HA: the ratio of SS is 3: 7.
(2) mixed solution of sericin and hydroxyapatite is put into ultrasonic cleaner, oscillation treatment 3～5min.
(3) taking out mixed solution, is 0.5% glutaraldehyde cross-linking agent to the concentration that wherein slowly drips trace, and magnetic stirring apparatus slowly stirs.
(4) mixed solution is moved into 96 porocyte culture plates fast, and put into immediately-20 ℃ of refrigerator precooling 12h.
(5) freezing 12h rear plate is taken out, at ambient temperature, rise again and process 4h, the sample of molding is taken out carefully.
(6) sample is put into again-20 ℃ of refrigerator and cooled and frozen, then put into-80 ℃ of freezer dryer lyophilizations, finally make sericin/hydroxyapatite composite porous bracket HA30.Its density is 39.7mg/ml, and porosity is 51.3%, and elastic modelling quantity is 10.56MPa, Fig. 3.
The preparation method of embodiment 7, a kind of sericin/hydroxyapatite composite porous bracket may further comprise the steps:
(1) a certain amount of sericin solution of accurate measuring places beaker, in proportion to wherein adding hydroxyapatite powder.Make HA: the ratio of SS is 5: 5.
(2) mixed solution of sericin and hydroxyapatite is put into ultrasonic cleaner, oscillation treatment 3～5min.
(3) taking out mixed solution, is 0.5% glutaraldehyde cross-linking agent to the concentration that wherein slowly drips trace, and magnetic stirring apparatus slowly stirs.
(4) mixed solution is moved into 96 porocyte culture plates fast, and put into immediately-20 ℃ of refrigerator precooling 12h.
(5) freezing 12h rear plate is taken out, at ambient temperature, rise again and process 4h, the sample of molding is taken out carefully.
(6) sample is put into again-20 ℃ of refrigerator and cooled and frozen, then put into-80 ℃ of freezer dryer lyophilizations, finally make sericin/hydroxyapatite composite porous bracket HA50.Its density is 52.7mg/ml, and porosity is 33%, and elastic modelling quantity is 15.64MPa, Fig. 4.
At last, it is also to be noted that what more than enumerate only is specific embodiments of the invention.Obviously, the invention is not restricted to above examples of implementation, many distortion can also be arranged.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention all should be thought protection scope of the present invention.
Claims (6)
1. the preparation method of a sericin/hydroxyapatite composite porous bracket is characterized in that may further comprise the steps:
(1) measures a certain amount of sericin solution and place beaker, in proportion to wherein adding hydroxyapatite powder, keep sericin consistent with the ratio of total amount in solution of hydroxyapatite; The weight ratio of described sericin and hydroxyapatite is 10: 1～1: 1;
(2) mixed solution of sericin and hydroxyapatite is put into ultrasonic cleaner, oscillation treatment 3～5min;
(3) taking out mixed solution, is 0.5% glutaraldehyde cross-linking agent to the concentration that wherein slowly drips trace, and magnetic stirring apparatus slowly stirs;
(4) mixed solution is moved into 96 porocyte culture plates fast, and put into immediately-20 ℃ of refrigerator precooling 12h;
(5) freezing 12h rear plate sample is taken out, under 20 ℃ of conditions, rise again and process 4h;
(6) flat samples after will rising again is again put into-20 ℃ of refrigerator and cooled and is frozen, and then puts into-80 ℃ of freezer dryer lyophilizations, makes the sericin/hydroxyapatite composite porous bracket of different proportion.
2. preparation method according to claim 1 is characterized in that: described sericin solution, and its preparation method may further comprise the steps:
(1) will remove Bombyxmori Linnaeus cocoon layer behind the pupa, and put into boiling water and carry out degumming process;
(2) behind the 30min, stop steaming and decocting, filter roguing;
(3) to put into Rotary Evaporators concentrated for the sericin solution after will filtering, and obtains the sericin solution of purpose concentration.
3. preparation method according to claim 1 is characterized in that: described hydroxylapatite powder, and its preparation method may further comprise the steps:
(1) gets saturated Ca (OH)
2Solution places 60 ℃ of water-baths, drips the H of 10%wt
3PO
4Solution, Ca (OH)
2Solution and H
3PO
4The volume ratio of solution is 70: 1～2, during continuous rapid stirring and keep water temperature;
(2) to drip the method for ammonia, the pH that adjusts solution remains on 8.7;
(3) drip H
3PO
4Continue to stir 3h behind the solution, stirring finishes to be placed on room temperature and leaves standstill 48h;
(4) the bottom precipitation is collected in the supernatant of inclining; Precipitation is cleaned 2-3 time with dehydrated alcohol first, cleans 3-4 time with distilled water again;
The precipitation that (5) will obtain is put into refrigerator and cooled and is frozen, and then moves in the freeze dryer dryly, grinds, and obtains hydroxyapatite powder.
4. preparation method according to claim 2 is characterized in that: the consumption of water is to the cocoon layer submergence in the described step (1).
5. preparation method according to claim 2 is characterized in that: after also comprising the sericin solution that obtains purpose concentration, extract sericin solution, carry out sds gel electrophoresis, measure the sericin molecular weight.
6. preparation method according to claim 3, it is characterized in that: wherein step (1) is to get the saturated Ca of 700mL (OH)
2Solution places 60 ℃ of water-baths, drips the H of 10%wt
3PO
4Solution 11mL, during use the continuous rapid stirring of magnetic stirring apparatus and keep water temperature.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010157889 CN101810881B (en) | 2010-04-27 | 2010-04-27 | Preparation method of sericin/hydroxyapatite composite porous bracket |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010157889 CN101810881B (en) | 2010-04-27 | 2010-04-27 | Preparation method of sericin/hydroxyapatite composite porous bracket |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101810881A CN101810881A (en) | 2010-08-25 |
| CN101810881B true CN101810881B (en) | 2013-01-02 |
Family
ID=42618340
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201010157889 Expired - Fee Related CN101810881B (en) | 2010-04-27 | 2010-04-27 | Preparation method of sericin/hydroxyapatite composite porous bracket |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101810881B (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102321270B (en) * | 2011-08-22 | 2012-12-12 | 陕西师范大学 | Preparation method of silk fibroin/hydroxyapatite/collagen composite porous stent |
| CN102526801B (en) * | 2012-02-20 | 2014-07-16 | 浙江大学 | Preparation method for flexible sericin protein/hydroxyapatite composite membrane |
| CN102532573B (en) * | 2012-02-20 | 2013-10-16 | 浙江大学 | Rapid molding method of sericin/hydroxyapatite composite membrane |
| CN103951831B (en) * | 2014-02-28 | 2016-08-17 | 华中科技大学同济医学院附属协和医院 | The preparation method and applications of sericin hydrogel |
| CN106512080A (en) * | 2016-11-24 | 2017-03-22 | 江苏爱西施科技服务咨询股份有限公司 | Preparation method for sericin protein hydrogel |
| CN110665055B (en) * | 2019-07-10 | 2022-08-26 | 赣南医学院第一附属医院 | Sericin/nano-hydroxyapatite tissue engineering bone graft and preparation method and application thereof |
| CN113372131B (en) * | 2021-04-26 | 2022-06-07 | 西安理工大学 | Micro-nano pore channel structure tetragonal phase BaTiO3Preparation method of/HA hollow microspheres |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101502672A (en) * | 2009-03-19 | 2009-08-12 | 浙江大学 | Method for preparing hydroxyapatite/silk fibroin compound porous stand material |
-
2010
- 2010-04-27 CN CN 201010157889 patent/CN101810881B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101502672A (en) * | 2009-03-19 | 2009-08-12 | 浙江大学 | Method for preparing hydroxyapatite/silk fibroin compound porous stand material |
Non-Patent Citations (4)
| Title |
|---|
| Akari Takeuchi et al..Heterogeneous nucleation of hydroxyapatite on protein: structural effect of silk sericin.《Journal of the Royal Society Interface》.2005,第2卷373-378. |
| Heterogeneous nucleation of hydroxyapatite on protein: structural effect of silk sericin;Akari Takeuchi et al.;《Journal of the Royal Society Interface》;20050713;第2卷;373-378 * |
| 类牙釉状丝胶蛋白/羟基磷灰石复合材料的合成及表征;金君等;《化学学报》;20091231;第67卷(第21期);2500-2504 * |
| 金君等.类牙釉状丝胶蛋白/羟基磷灰石复合材料的合成及表征.《化学学报》.2009,第67卷(第21期),2500-2504. |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101810881A (en) | 2010-08-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101810881B (en) | Preparation method of sericin/hydroxyapatite composite porous bracket | |
| CN101502672B (en) | Method for preparing hydroxyapatite/silk fibroin compound porous stand material | |
| Volkov et al. | Poly (3-hydroxybutyrate)/hydroxyapatite/alginate scaffolds seeded with mesenchymal stem cells enhance the regeneration of critical-sized bone defect | |
| Peng et al. | Preparation and evaluation of porous chitosan/collagen scaffolds for periodontal tissue engineering | |
| CN104117098B (en) | A kind of magnetic hydroxylapatite/polymer three-dimensional porous support materials with alignment magnetic field and preparation method thereof | |
| Zhang et al. | Preparation and biocompatibility evaluation of apatite/wollastonite-derived porous bioactive glass ceramic scaffolds | |
| CN101890184B (en) | Cartilage derived collagen sponge scaffold and preparation method thereof | |
| PT2117617E (en) | A collagen/hydroxyapatite composite scaffold, and process for the production thereof | |
| CN104001208B (en) | Preparation method for biomacromolecule/chitin nanocrystalline composite scaffold material | |
| CN101549171A (en) | II type collagen sponge bracket and uses thereof | |
| CN101773684A (en) | Preparation method of fibroin/hydroxyapatite porous scaffold | |
| CN110818921A (en) | Rapidly-curable double-crosslinked hydrogel and preparation method and application thereof | |
| CN102973984A (en) | Preparation method and application of composite porous scaffold | |
| CN105664260A (en) | Method for preparing bone tissue engineering three-dimensional porous support based on graphene/silk fibroin | |
| CN1954890A (en) | Preparation method of tissue engineering bone cartilage compound and its application | |
| Yang et al. | Gelatin-assisted conglutination of aligned polycaprolactone nanofilms into a multilayered fibre-guiding scaffold for periodontal ligament regeneration | |
| CN102085392A (en) | Nano-apatite/collagen compound stent and preparation method and application thereof | |
| CN104497327A (en) | Fibroin hyaluronic acid composite membrane with hygroscopic property and without cross-linking agent and preparation | |
| Wibowo et al. | Development of salt leached silk fibroin scaffold using direct dissolution techniques for cartilage tissue engineering | |
| CN113583455A (en) | Collagen-modified chitosan double-network hydrogel, biological ink, preparation method and application | |
| CN107929814A (en) | A kind of calcium sulfate/hydroxyapatite crystal whisker Composite Bone material for repairing and preparation method thereof | |
| Sah et al. | Eggshell membrane protein modified silk fibroin-poly vinyl alcohol scaffold for bone tissue engineering: in vitro and in vivo study | |
| CN112138216A (en) | Hybrid cross-linked skull repairing porous membrane with highly bionic bone matrix and preparation method thereof | |
| CN114854053B (en) | Polyethylene glycol-chitosan double-network hydrogel and preparation method and application thereof | |
| CN103920187B (en) | The bone renovating material that a kind of fibroin albumen and decalcified bone matrix are compound |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20130102 Termination date: 20210427 |