CN101690830B - Preparation method of bionic cartilage extracellular matrix for tissue engineering - Google Patents
Preparation method of bionic cartilage extracellular matrix for tissue engineering Download PDFInfo
- Publication number
- CN101690830B CN101690830B CN 200910191034 CN200910191034A CN101690830B CN 101690830 B CN101690830 B CN 101690830B CN 200910191034 CN200910191034 CN 200910191034 CN 200910191034 A CN200910191034 A CN 200910191034A CN 101690830 B CN101690830 B CN 101690830B
- Authority
- CN
- China
- Prior art keywords
- extracellular matrix
- tissue engineering
- bionic
- cartilage
- cartilage extracellular
- 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.)
- Active
Links
Abstract
The invention discloses a preparation method of a bionic cartilage extracellular matrix for tissue engineering, which comprises the following steps of: selecting a raw material having the same main components as the normal articular cartilage extracellular matrix as a bionic raw material to prepare the bionic cartilage extracellular matrix for the tissue engineering; fully dissolving the selected bionic raw material in organic acid; and then carrying out hole making processing, curing processing and strengthening processing in sequence to the selected bionic raw material. The method makes the material structure of the finally obtained bionic cartilage extracellular matrix for the tissue engineering have physical structure characteristics suitable for biological behavior requirements such as vaccination, survival, multiplication and the like of a cartilage seed cell and have good biocompatibility, appropriate degradation rate and biomechanical strength at the same time. The preparation method has the advantages of wide material source, low cost, simple and easy process and good reproducibility, and the prepared product can be widely used to repair a large area of articular cartilage defect and has fewer clinical complications.
Description
Technical field
The present invention relates to the fabrication of cartilage extracellular matrix, particularly a kind of method for preparing of bionic cartilage extracellular matrix for tissue engineering.
Background technology
Clinical common by wound or osteopathia joint caused position cartilage defect, have a strong impact on patients ' life quality, become the one of the main reasons of present limbs disability.U.S.'s sickness rate is 1.5 ‰～3 ‰, and China is about 5～6 times of the U.S., and is ascendant trend year by year.Articular cartilage belongs to hyaline cartilage, lacks neural blood vessel nutrition, and the damage back self is difficult to repair.All there is open defect in clinical existing treatment measure, and its expectant treatment and joint debridement art can only respite pain, can not stop the development of the course of disease; The bone and cartilage autotransplantation art can cause and supply district's damage, and it is limited to originate, and it is damaged to be difficult to repair larger area; The allograph bone cartilage grafting exists immunological rejection and pathophorous possibility; Artificial joint replacement then expensive, complication is more, revision rate is higher, and is particularly big to the influence of the body and mind of young patient, financial burden is heavy.
The birth of tissue engineering and fast development thereof provide new technique for the Regeneration and Repair of articular cartilage.The eighties in 20th century, two stages were experienced in the research of tissue engineering along with the deep development of basic subjects such as cytobiology and Engineering Materials: preceding 10 years mainly is to confirm to utilize cell and biomaterial to make up the feasibility of tissue; The back then deepened continuously at aspects such as research contents, means, technological improvement and clinic trial in more than 10 year, prepared with the expansion tissue engineering product for improving curative effect.Sweden scholar in 1987 reports that transplanting the amplification in vitro chondrocyte comes clinical treatment from the body articular cartilage defect.The tissue engineering product Carticel
of the FDA of FDA (Food and Drug Adminstration) approval Genzyme company in 1997 gets into clinical, has surpassed 4000 patients up till now and has benefited from this product.The tissue engineering bone/cartilage that Chinese People's Armed Police hospital general in 2004 makes up German Verigen company is applied to clinical, is applied to clinical but China also lacks the tissue engineering bone/cartilage host material with independent intellectual property right at present.
Tissue engineering bracket material helps sticking, breed and even breaking up of cell for making up the three-dimensional rack that cells of tissues provides, for the cell growth provides suitable external environment.In organizational project, timbering material plays the effect of extracellular matrix, is 26S Proteasome Structure and Function bionical of pair cell epimatrix.It not only plays a supportive role, and keeps former shape in a organized way, but also plays template action, for cell provides the place of the boarding of relying, growth, differentiation and propagation, thereby guides the regeneration of damaged tissues and the structure of control regenerating tissues.
Research shows that the natural joint cartilage is made up of chondrocyte and extracellular matrix, and both cooperation have guaranteed the normal physiological function of articular cartilage.Chondrocyte accounts for 5% of whole tissue volume, and major function is secretion extracellular matrix, the renewal that keeps substrate.Extracellular matrix 70% is made up of water, and all the other are made up of collagen and proteoglycan, mainly is responsible for carrying chondrocyte, regulates the propagation and the differentiation of cell, buffering joint stress.Collagen accounts for the 60%-80% of cartilage dry weight, and wherein 95% is the II Collagen Type VI.The proteoglycan polymer accounts for the 20%-40% of hyaline cartilage dry weight, mainly comprises chondroitin sulfate and hyaluronic acid, and both combine to form the proteoglycan polymer.GAG residue in the polymer has negative charge, and intermolecular mutual repulsion has intensive hydrophilic, and this is the major reason that cartilage matrix keeps large quantity of moisture.Key between chondroitin sulfate and the collagen fiber connects, and has avoided the substrate overly hydrated, and then has prevented chondromalacia.No matter the tissue engineering bone/cartilage timbering material of prior art for preparing and normal cartilage matrix all exist significant difference on the composition or on the structure.
Summary of the invention
In view of this; The present invention provides a kind of method for preparing of bionic cartilage extracellular matrix for tissue engineering; Can make with like normal cartilage structure and the constituent class, the bionic cartilage extracellular matrix for tissue engineering that bionical degree is high, and this bionic cartilage extracellular matrix for tissue engineering has excellent biological compatibility, degradable absorbability and mechanical property; Simultaneously with low cost, become the desirable biomaterial of repairing cartilage defect and the needs that satisfy clinical extensive application.
The method for preparing of bionic cartilage extracellular matrix for tissue engineering of the present invention: may further comprise the steps:
A. choose the bionical raw material of preparation bionic cartilage extracellular matrix for tissue engineering, said bionical raw material (wt%) by mass percentage comprises following composition: 60～80 II collagen type, 15～30 chondroitin sulfate and 5～10 hyaluronic acid;
B. obtain acid solution after using concentration fully to dissolve as the hydrochloric acid of 0.01mol/L～0.1mol/L the bionical raw material of selecting for use among the step a as the organic acid of 0.01mol/L～0.1mol/L or concentration;
C. with the acid solution that obtains among the step b successively through drilling processing, cured and intensive treatment and obtain the bionic cartilage extracellular matrix for tissue engineering that required suitable cartilage seed cell biological behaviour requires.
Further, the bionic cartilage extracellular matrix for tissue engineering that obtains among the step c possesses following physical arrangement parameter: aperture 100-150 μ m, porosity 60-90%, hole passband 100%;
Further, the organic acid described in the step b is acetic acid or malonic acid;
Further; The described drilling of step c is treated to: under 20-25 ℃ condition; Perforating agent is added in the acid solution that step b obtains; Fully stir and make that to obtain bionical raw material total mass concentration behind its mix homogeneously be 5%～30% bionic cartilage extracellular matrix for tissue engineering solution, more described bionic cartilage extracellular matrix for tissue engineering solution is injected the mould that is used for making bionic cartilage extracellular matrix for tissue engineering for preparing in advance, to mould pressurization, sealing and in-10 ℃～-30 ℃ environment, place 2-4h and get final product; Wherein, perforating agent is the water solublity perforating agent of particle diameter at 100-150 μ m;
Further, the described cured of step c is freezing lyophilizing, and the intensive treatment described in the step c is chemical crosslinking;
Further, said cured is: with among the step c after the acid solution that drilling is handled places-70 ℃～-90 ℃ environment to place 6-12h, carry out lyophilizing and obtain freeze dried composite;
Further, said intensive treatment comprises the steps: 1. said freeze dried composite to be taken out and is soaked in the cross-linking agent solution that concentration is 5-10%, and every 2-4h changes cross-linking agent solution; Behind the 24h cross-linking agent solution that contains composite is carried out low-temperature freeze drying and obtains compound matrix material; 2. with compound matrix material through deionized water repeatedly rinsing to compound matrix material be neutral, obtain porous compound matrix material, 3. porous compound matrix material is carried out lyophilizing; Again through the X-radiation sterilization; Can obtain required bionic cartilage extracellular matrix for tissue engineering product, wherein cross-linking agent is aldehydes cross-linking agent, carbonization two imido class cross-linking agent, genipin or 1, and 4-two (3; The 4-hydroxy benzenes)-2,3-dimethylbutane (NDGA).
The present invention also provides the preferred implementation method of following preparation bionic cartilage extracellular matrix for tissue engineering, comprises the steps:
1) choose the bionical raw material for preparing bionic cartilage extracellular matrix for tissue engineering, (wt%) comprises following composition by mass percentage:
The II collagen type of 60-80, the chondroitin sulfate of 15-30, the hyaluronic acid of 5-10;
2) be dissolved in II collagen type, chondroitin sulfate and the hyaluronic acid of the said amount of step 1) in acetic acid, malonic acid solvent or the hydrochloric acid that concentration is 0.01mol/L～0.1mol/L and fully stir and it is dissolved fully obtain acid solution;
3) under 20-25 ℃ of condition; With particle diameter be the water solublity perforating agent of 100-150 μ m will the amount of asking for according to designed porosity and add acid solution after; Fully stir that to make its mix homogeneously obtain bionical raw material total mass concentration be 5%～30% bionic cartilage extracellular matrix for tissue engineering solution; Wherein, perforating agent is preferably NaCl or KCl crystal;
4) the bionic cartilage extracellular matrix for tissue engineering solution with the step 3) gained injects the also pressurization of mould, the sealing that are used for making bionic cartilage extracellular matrix for tissue engineering for preparing in advance;
5) placing-70 ℃ again after placing-10 ℃～-30 ℃ environment to place 2-4h on the mould of the injection acid solution described in the step 4)--90 ℃ environment is placed 6-12h, carries out lyophilizing at last again;
6) according to the collagen cross-linking specification requirement, freeze dried composite in the step 5) is taken out, be soaked in the cross-linking agent solution that concentration is 5-10%; Every 2-4h changes cross-linking agent solution; Behind the crosslinked 24h composite is carried out low-temperature freeze drying and obtain compound matrix material, wherein cross-linking agent is preferably glutaraldehyde, genipin (Genipin) or 1, and 4-two (3; The 4-hydroxy benzenes)-2,3-dimethylbutane (NDGA);
7) with freeze dried compound matrix material in the step 6) use deionized water repeatedly rinsing to compound matrix material be neutrality, obtain porous compound matrix material;
8) said porous compound matrix material is carried out low-temperature freeze drying; After the X-radiation sterilization, can obtain the bionic cartilage extracellular matrix for tissue engineering product again; Said bionic cartilage extracellular matrix for tissue engineering product possesses following physical arrangement parameter: the aperture: 100～150 μ m; Porosity 60～90%, hole passband 100%.
Beneficial effect of the present invention is: the method for preparing of bionic cartilage extracellular matrix for tissue engineering of the present invention; Choose and the bionical raw material of human body natural joint cartilage cell epimatrix main component identical materials as the preparation bionic cartilage extracellular matrix for tissue engineering; In the preparation process; Successively through drilling processing, cured and intensive treatment, make the bionic cartilage extracellular matrix for tissue engineering that finally obtains possess the physical arrangement characteristic that biological behaviours such as the inoculation of suitable cartilage seed cell, survival and propagation require after the bionical raw material of choosing fully dissolved through organic acid, possess good biocompatibility, suitable degradation rate and good biomechanical strength simultaneously; And; Used bionical raw material wide material sources, with low cost, simple for process; Favorable reproducibility, the bionic cartilage extracellular matrix for tissue engineering that makes can be widely used in repairing large-area articular cartilage defect and clinical complication few.
Other advantages of the present invention, target and characteristic will be set forth in description subsequently to a certain extent; And to a certain extent; Based on being conspicuous to those skilled in the art, perhaps can from practice of the present invention, obtain instruction to investigating of hereinafter.
The specific embodiment
Below will carry out detailed description to the preferred embodiments of the present invention.Should be appreciated that preferred embodiment has been merely explanation the present invention, rather than in order to limit protection scope of the present invention.
Embodiment 1
The method for preparing of the bionic cartilage extracellular matrix for tissue engineering of present embodiment may further comprise the steps:
II collagen type, chondroitin sulfate and hyaluronic acid are dissolved in the 0.05mol/L malonic acid solution according to mass percent at 6: 3: 1, fully stir it is disperseed fully, be mixed with bionical raw material total mass concentration and be 15% acid collagen mixed liquid of protein; According to the tissue engineering principle, selecting granularity for use is that the NaCl crystal of 125-150 μ m is as perforating agent; Get 100ml acid collagen mixed liquid of protein, under 20 ℃ condition, adding particle diameter is the NaCl crystal 100g of 125-150 μ m, fully stirs and makes its mix homogeneously; Getting acid collagen mixed liquid of protein that 10ml contains NaCl, to inject the diameter be used for making bionic cartilage extracellular matrix for tissue engineering be the 3cm cylindrical die, mould pressurizeed, seals and place-10 ℃ of refrigerators place 4h; And then place-70 ℃ of refrigerators to place 6h the mould.Place the low-temperature freeze-drying machine lyophilizing at last; Freeze dried collagen protein, chondroitin sulfate, hyaluronic acid composite are taken out from mould again, be immersed in concentration and be in 5% the genipin solution, every 4h changes primary cross-linking agent solution, low-temperature freeze drying behind the crosslinked 24h; At last with freeze dried compound matrix material with rinsed with deionized water 5 times (each 20mins) after; With the neutral porous compound matrix material of gained low-temperature freeze drying once more; After the X-radiation sterilization obtains required tissue engineering bone/cartilage bionic extracellular matrix product, product is sealed low temperature storage get final product.
In the present embodiment, drill process is a prior art, and perforating agent is preferably the NaCl crystal for possessing specified particle size and certain water miscible crystal, certainly, also can be preferably the KCl crystal, makes the porosity of product high, and distribution consistency degree is good; Cured is employed in freezing lyophilizing under-70 ℃ the condition, ensures that material can degeneration or inactivation, and intensive treatment adopts chemical crosslinking; In the present embodiment, cross-linking agent is that concentration is 5% genipin, the compound matrix material that obtains through intensive treatment; Its biomechanical strength is high, and clinical ruggedness is good.
Through a large amount of experiment confirms of research worker of the present invention, crosslinker concentration is that 5-10%, kind are aldehydes cross-linking agent, carbonization two imido class cross-linking agent or 1, and 4-two (3; The 4-hydroxy benzenes)-2,3-dimethylbutane (NDGA) all can be realized the object of the invention, and; A large amount of experiment confirms through research worker of the present invention; In this method, being used for the dissolved solvent of bionical raw material is that the organic acid of 0.01mol/L～0.1mol/L can be realized the object of the invention equally for the concentration of other kinds, and organic acid to adopt concentration be acetic acid or the malonic acid of 0.01mol/L～0.1mol/L; Can make in the preparation process; The foreign ion kind of bringing solution into is few, is convenient to the separation and the removal of foreign ion in the subsequent technique, certainly; In the present embodiment, the hydrochloric acid that is used for the dissolved solvent of bionical raw material and is concentration and be 0.01mol/L～0.1mol/L can be realized the object of the invention equally.
With the tissue engineering bone/cartilage bionic extracellular matrix product of the inventive method preparation, can be widely used in the external structure or the internal regeneration of cartilage, it is good to be used to repair large-area articular cartilage defect clinical effectiveness, and complication is few, and is simultaneously with low cost, makes easily.
According to the ISO10993 series standard, adopt methods such as scanning electron microscopic observation, Experiments of Machanics, degradation test, cell toxicity test, genetic toxicity test that the physicochemical properties such as morphosis, mechanical property, biological safety and degraded and absorbed property of tissue engineering bone/cartilage bionic extracellular matrix product are detected.Through scanning electron microscopic observation, visible aperture is 125-150 μ m; Porosity is 90%; The hole passband is 100%.Testing result proves that tissue engineering bone/cartilage bionic extracellular matrix product of the present invention has the interior spatial structure and the proportion of composing of designing requirement; Have the favorable tissue compatibility and biological safety; Be beneficial to cell adhesion, hypertrophy, have excellent mechanical intensity, can satisfy the mechanics requirement of implant site; Have the controlled degradation absorbability, can make through artificial regulatory that the cambium speed of growth is complementary in its degraded and absorbed speed and the body.
Adopt this tissue engineering bone/cartilage bionic extracellular matrix product to combine seed cell technology of preparing, two-phase gel inoculation technique and piece of tissue In vitro culture technique construction through engineering approaches cartilaginous tissue; Large animal (pig) body is implanted into to be repaired experimental result and shows that the through engineering approaches cartilaginous tissue that adopts tissue engineering bone/cartilage bionic extracellular matrix product of the present invention the to make up back that implants is fully integrated with normal surrounding tissue, degradation time and cambium speed of growth coupling on every side; The repair of cartilage tissue has the biological characteristics of similar natural joint cartilage.
Embodiment 2
The method for preparing of the bionic cartilage extracellular matrix for tissue engineering of present embodiment may further comprise the steps:
II collagen type, chondroitin sulfate and hyaluronic acid are dissolved in the 0.05mol/L malonic acid solution according to 7: 2.5: 0.5 mass percent of mass percent; Fully stir it is disperseed fully, be mixed with bionical raw material total mass concentration and be 5% acid collagen mixed liquid of protein; Again according to the tissue engineering principle, selecting granularity for use is that the KCl crystal of 100-125 μ m is as perforating agent; 100ml acid collagen mixed liquid of protein again, under 25 ℃ condition, adding particle diameter is the KCl crystal grain 120g of 100-125 μ m, fully stirs and makes its mix homogeneously; Getting acid collagen mixed liquid of protein that 10ml contains KCl, to inject diameter be the 3cm cylindrical die, mould pressurizeed, seals and places-20 ℃ refrigerator and place 2h; And then place-80 ℃ refrigerator to place 9h the mould, place the low-temperature freeze-drying machine lyophilizing at last; Freeze dried collagen protein, chondroitin sulfate, hyaluronic acid composite are taken out from mould again, be immersed in concentration and be in 10% the NDGA solution, every 2h changes primary cross-linking agent solution, low-temperature freeze drying behind the crosslinked 24h; At last with freeze dried compound matrix material with rinsed with deionized water 5 times (each 20mins); Again with the neutral porous compound matrix material of gained low-temperature freeze drying once more; After the X-radiation sterilization obtains required tissue engineering bone/cartilage bionic extracellular matrix product, product is sealed low temperature storage get final product.
According to the ISO10993 series standard, adopt methods such as scanning electron microscopic observation, Experiments of Machanics, degradation test, cell toxicity test, genetic toxicity test that the physicochemical properties such as morphosis, mechanical property, biological safety and degraded and absorbed property of tissue engineering bone/cartilage bionic extracellular matrix product are detected.Through scanning electron microscopic observation, visible aperture is 100-125 μ m; Porosity is 80%; The hole passband is 100%.Testing result proves that tissue engineering bone/cartilage bionic extracellular matrix product of the present invention has the interior spatial structure and the proportion of composing of designing requirement; Have the favorable tissue compatibility and biological safety, be beneficial to cell adhesion, hypertrophy; Have excellent mechanical intensity, can satisfy the mechanics requirement of implant site; Have the controlled degradation absorbability, can make through artificial regulatory that the cambium speed of growth is complementary in its degraded and absorbed speed and the body.
Adopt this tissue engineering bone/cartilage bionic extracellular matrix product to combine seed cell technology of preparing, two-phase gel inoculation technique and piece of tissue In vitro culture technique construction through engineering approaches cartilaginous tissue; Large animal (pig) body is implanted into to be repaired experimental result and shows that the through engineering approaches cartilaginous tissue that adopts tissue engineering bone/cartilage bionic extracellular matrix product of the present invention the to make up back that implants is fully integrated with normal surrounding tissue, degradation time and cambium speed of growth coupling on every side; The repair of cartilage tissue has the biological characteristics of similar natural joint cartilage.
Embodiment 3
The method for preparing of the bionic cartilage extracellular matrix for tissue engineering of present embodiment may further comprise the steps:
II collagen type, chondroitin sulfate and hyaluronic acid are dissolved in the 0.1mol/L acetum according to 8: 1.5: 0.5 mass percent of mass percent; Fully stir it is disperseed fully, be mixed with bionical raw material total mass concentration and be 30% acid collagen mixed liquid of protein; Again according to the aperture design requirement of bionic cartilage extracellular matrix for tissue engineering, selecting granularity for use is that the NaCl crystal of 120-140 μ m is as perforating agent; Get 100ml acid collagen mixed liquid of protein, under 22 ℃ condition, adding particle diameter is the NaCl crystal grain 90g of 120-140 μ m, fully stirs and makes its mix homogeneously; Getting acid collagen mixed liquid of protein that 10ml contains NaCl, to inject diameter be the 3cm cylindrical die, mould pressurizeed, seals and place-30 ℃ of refrigerators place 3h; And then place-90 ℃ of refrigerators to place 12h the mould, place the low-temperature freeze-drying machine lyophilizing at last; Freeze dried collagen protein, chondroitin sulfate, hyaluronic acid composite are taken out from mould again, be immersed in concentration and be in 8% the glutaraldehyde solution, every 3h changes primary cross-linking agent solution, low-temperature freeze drying behind the crosslinked 24h; At last with freeze dried compound matrix material with rinsed with deionized water 5 times (each 20mins); Again with the neutral porous compound matrix material of gained low-temperature freeze drying once more; After the X-radiation sterilization obtains required tissue engineering bone/cartilage bionic extracellular matrix product, product is sealed low temperature storage get final product.
According to the ISO10993 series standard, adopt methods such as scanning electron microscopic observation, Experiments of Machanics, degradation test, cell toxicity test, genetic toxicity test that the physicochemical properties such as morphosis, mechanical property, biological safety and degraded and absorbed property of tissue engineering bone/cartilage bionic extracellular matrix product are detected.Through scanning electron microscopic observation, visible aperture is 120-140 μ m; Porosity is 60%; The hole passband is 100%.Testing result proves that tissue engineering bone/cartilage bionic extracellular matrix product of the present invention has the interior spatial structure and the proportion of composing of designing requirement; Have the favorable tissue compatibility and biological safety, be beneficial to cell adhesion, hypertrophy; Have excellent mechanical intensity, can satisfy the mechanics requirement of implant site; Have the controlled degradation absorbability, can make through artificial regulatory that the cambium speed of growth is complementary in its degraded and absorbed speed and the body.
Adopt this tissue engineering bone/cartilage bionic extracellular matrix product to combine seed cell technology of preparing, two-phase gel inoculation technique and piece of tissue In vitro culture technique construction through engineering approaches cartilaginous tissue; Large animal (pig) body is implanted into to be repaired experimental result and shows that the through engineering approaches cartilaginous tissue that adopts tissue engineering bone/cartilage bionic extracellular matrix product of the present invention the to make up back that implants is fully integrated with normal surrounding tissue, degradation time and cambium speed of growth coupling on every side; The repair of cartilage tissue has the biological characteristics of similar natural joint cartilage.
Explanation is at last; Above embodiment is only unrestricted in order to technical scheme of the present invention to be described; Although with reference to preferred embodiment the present invention is specified, those of ordinary skill in the art should be appreciated that and can make amendment or be equal to replacement technical scheme of the present invention; And not breaking away from the aim and the scope of technical scheme of the present invention, it all should be encompassed in the middle of the claim scope of the present invention.
Claims (1)
1. the method for preparing of a bionic cartilage extracellular matrix for tissue engineering is characterized in that: comprise the steps:
1) choose the bionical raw material for preparing bionic cartilage extracellular matrix for tissue engineering, (wt%) is grouped into by following one-tenth by mass percentage:
The II collagen type of 60-80, the chondroitin sulfate of 15-30, the hyaluronic acid of 5-10;
2) be dissolved in II collagen type, chondroitin sulfate and the hyaluronic acid of the said amount of step 1) in acetic acid, malonic acid solvent or the hydrochloric acid that concentration is 0.01mol/L～0.1mol/L and fully stir and it is dissolved fully obtain acid solution;
3) under 20-25 ℃ of condition; With particle diameter be the water solublity perforating agent of 100-150 μ m will the amount of asking for according to designed porosity and add acid solution after; Fully stir that to make its mix homogeneously obtain bionical raw material total mass concentration be 5%～30% bionic cartilage extracellular matrix for tissue engineering solution; Wherein, perforating agent is NaCl or KCl crystal;
4) the bionic cartilage extracellular matrix for tissue engineering solution with the step 3) gained injects the also pressurization of mould, the sealing that are used for making bionic cartilage extracellular matrix for tissue engineering for preparing in advance;
5) place-70 ℃～-90 ℃ environment to place 6-12h again after placing-10 ℃～-30 ℃ environment to place 2-4h on the mould of the injection acid solution described in the step 4), carry out lyophilizing at last again;
6) according to the collagen cross-linking specification requirement, freeze dried composite in the step 5) is taken out, be soaked in the cross-linking agent solution that concentration is 5-10%; Every 2-4h changes cross-linking agent solution; Behind the crosslinked 24h composite is carried out low-temperature freeze drying and obtain compound matrix material, wherein cross-linking agent glutaraldehyde, genipin or 1,4-two (3; The 4-hydroxy benzenes)-2,3-dimethylbutane;
7) with freeze dried compound matrix material in the step 6) use deionized water repeatedly rinsing to compound matrix material be neutrality, obtain porous compound matrix material;
8) said porous compound matrix material is carried out low-temperature freeze drying; After the X-radiation sterilization, can obtain the bionic cartilage extracellular matrix for tissue engineering product again; Said bionic cartilage extracellular matrix for tissue engineering product possesses following physical arrangement parameter: the aperture: 100～150 μ m; Porosity 60～90%, hole passband 100%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200910191034 CN101690830B (en) | 2009-09-29 | 2009-09-29 | Preparation method of bionic cartilage extracellular matrix for tissue engineering |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200910191034 CN101690830B (en) | 2009-09-29 | 2009-09-29 | Preparation method of bionic cartilage extracellular matrix for tissue engineering |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101690830A CN101690830A (en) | 2010-04-07 |
| CN101690830B true CN101690830B (en) | 2012-12-05 |
Family
ID=42079577
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200910191034 Active CN101690830B (en) | 2009-09-29 | 2009-09-29 | Preparation method of bionic cartilage extracellular matrix for tissue engineering |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101690830B (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101810855B (en) * | 2010-05-20 | 2012-06-13 | 广州创尔生物技术有限公司 | II-type collagen joint cartilage fluid and preparation method thereof |
| CN102344905B (en) * | 2010-08-04 | 2014-04-23 | 北京大学 | Bionic culture model for chondrocyte, and preparation method thereof |
| CN101934092A (en) * | 2010-08-27 | 2011-01-05 | 中国人民解放军第三军医大学第一附属医院 | Injection-type cartilage bionic matrix for regenerative repair of cartilage and method for using same |
| CN102949751A (en) * | 2012-11-28 | 2013-03-06 | 川北医学院第二临床医学院 | Preparation method for tissue engineering collagen-hyaluronic acid-chondroitin sulfate electrostatic spinning bracket |
| CN111705034A (en) * | 2020-06-30 | 2020-09-25 | 山东省医药生物技术研究中心（山东省病毒研究所） | Method for efficiently promoting chondrocyte proliferation |
| CN114908038B (en) * | 2022-06-06 | 2024-03-15 | 苏州大学 | In-vitro 3D joint injury model and construction and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1319436A (en) * | 2001-02-28 | 2001-10-31 | 中国医学科学院生物医学工程研究所 | Compound collagen stroma tissue engineering support and preparation method thereof |
| EP1312383A2 (en) * | 2001-11-20 | 2003-05-21 | Ed Geistlich Söhne AG Für Chemische Industrie | Resorbable extracellular matrix containing collagen I and collagen II for reconstruction of cartilage |
| CN1836740A (en) * | 2006-04-21 | 2006-09-27 | 华南理工大学 | Composite tri-dimensional porous bone tissue engineering stent material and its preparation method and uses |
-
2009
- 2009-09-29 CN CN 200910191034 patent/CN101690830B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1319436A (en) * | 2001-02-28 | 2001-10-31 | 中国医学科学院生物医学工程研究所 | Compound collagen stroma tissue engineering support and preparation method thereof |
| EP1312383A2 (en) * | 2001-11-20 | 2003-05-21 | Ed Geistlich Söhne AG Für Chemische Industrie | Resorbable extracellular matrix containing collagen I and collagen II for reconstruction of cartilage |
| CN1836740A (en) * | 2006-04-21 | 2006-09-27 | 华南理工大学 | Composite tri-dimensional porous bone tissue engineering stent material and its preparation method and uses |
Non-Patent Citations (1)
| Title |
|---|
| 阎继红等.胶原-透明质酸-硫酸软骨素复合三维支架体外构建组织工程软骨的实验研究.《中国修复重建外科杂志》.2006,第20卷(第2期),130-133. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101690830A (en) | 2010-04-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Zhang et al. | Three-dimensional gelatin and gelatin/hyaluronan hydrogel structures for traumatic brain injury | |
| CN101690830B (en) | Preparation method of bionic cartilage extracellular matrix for tissue engineering | |
| CN101366978B (en) | Fine particle tissue filling material for injection and preparation method thereof | |
| CN101020082B (en) | Bone repairing material and its prepn process and use | |
| CN105169486B (en) | A kind of nerve repair material of combination Acellular nerve application | |
| AU2009266859A1 (en) | Compositions and methods for tissue filling and regeneration | |
| CN102380129B (en) | Sodium hyaluronate and KGM porous bracket material and method for preparing same | |
| CN102552981A (en) | Method for preparing tissue-engineered bone/cartilage integrated scaffold | |
| CN105246495A (en) | Decellularized biomaterial form non-mammalian tissue | |
| CN106492281B (en) | Biocompatible bone graft and preparation method thereof | |
| CN103007358B (en) | Cartilage tissue engineering fiber scaffold material and preparation method thereof | |
| CN106075588A (en) | A kind of bioceramic gel complex material substituting reparation for cartilage and its preparation method and application | |
| CN102727935A (en) | Preparation method and device of duramater/spinal dural transplanting substitute | |
| CN108201632A (en) | A kind of articular cartilage repaiies scaffold | |
| CN108670946A (en) | Treat Cellular gels preparation of articular cartilage damage and application thereof and the active gel solution of holding freeze-stored cell used | |
| Bhat et al. | Biomaterials in regenerative medicine | |
| CN101856515B (en) | Method for preparing artificial bone from chitosan and shell powder serving as raw materials | |
| CN109182249B (en) | Preparation method of scaffold material for cell transplantation for in vivo repair | |
| CN102895702A (en) | Composite manual bile duct and preparation method thereof | |
| CN106730011A (en) | A kind of xenogenesis acellular nerve graft thing with prevention tissue adhesion function and preparation method thereof | |
| CN112076350B (en) | Biomimetic mineralized hydrogel with nano-micron composite structure and high mineral density as well as preparation method and application thereof | |
| CN106075567A (en) | A kind of gel biological pottery cartilage implant substituting reparation for cartilage and its preparation method and application | |
| JP3839777B2 (en) | Use of a biological material containing a three-dimensional skeleton of a hyaluronic acid derivative for preparing a graft in arthroscopy, and an instrument kit for transplanting said biological material by arthroscopy | |
| CN108042567A (en) | For the life assemblage preparation of repair of cartilage and its application | |
| CN209519145U (en) | Needle holder being applied to surgery Minimally nerve trachea |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |