CN1319436A - Compound collagen stroma tissue engineering support and preparation method thereof - Google Patents
Compound collagen stroma tissue engineering support and preparation method thereof Download PDFInfo
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- CN1319436A CN1319436A CN 01109205 CN01109205A CN1319436A CN 1319436 A CN1319436 A CN 1319436A CN 01109205 CN01109205 CN 01109205 CN 01109205 A CN01109205 A CN 01109205A CN 1319436 A CN1319436 A CN 1319436A
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Abstract
The present invention belongs to preparation of novel compound collagen base tissue engineering scaffold, and its composition contains 10 portions of collagen, 1-5 portions of chondriotin sulfate, 0.5-3 portions of chitin and 0.5-2 portions of polyvinyl alcohol. The collagen swelling liquor and chitin and polyvinyl alcohol are mixed together, and freeze-dried to obtain said scaffold, the said scaffold is immersed in the crosslinking solution containing chondroitin sulfate, cross-linked, freeze-dried and sterilized so as to obtain the invented product. It possesses good biological compatibility, can premote cell adhesion and multiplication, and its cost is low.
Description
The invention belongs to the preparation of tissue engineering bracket.
Collagen and mucopolysaccharide material all are the n cell epimatrixs, use extensivelyr in the research aspect tissue engineering bracket, and it is the preferred material that makes up organizational project organs such as artificial skin, cartilage, blood vessel.But present applied timbering material performance is also unsatisfactory.
The purpose of this invention is to provide a kind of compound collagen stroma tissue engineering support and preparation method thereof, the present invention is used for the tissue engineering bracket that soft tissue cells is cultivated, this support has improved the cell adhesion performance of existing collagen-based materials, has reduced the cytotoxicity of material and has improved the preparation technology of timbering material.
Weight of the present invention is formed: 10 parts of collagens, chondroitin sulfate 1-5 part, chitosan 0.5-3 part, polyvinyl alcohol 0.5-2 part.
The preparation method of compound collagen stroma tissue engineering support comprises the steps:
(1) be the collagen malonic acid swelling solution of 0.4-1.0% by metering configuration solids content, acetum dissolving chitosan with 0.2M, 0.3% malonic acid solution dissolve polyvinyl alcohol, and with in the molten account liquid of its adding collagen, stirring obtains collagen-chitin-poly-vinyl alcohol solution;
(2) the centrifugal bubble of removing places the lyophilizing container, and control cryogenic temperature-30 ℃ obtains porous collagen-chitin-polyvinyl alcohol timbering material to-10 ℃;
(3) from self-conceit pipe cartilage, extract chondroitin sulfate with the papain digestion method; Concentration is dissolved in the crosslinker solution for the 0.1-0.5% chondroitin sulfate, and crosslinker solution is the 20-40% alcoholic solution that contains the 2N-morphine quinoline-ethyl sulfonic acid (MES) of 1-ethyl-3-3-dimethylaminopropyl-carbonization two imido (EDC);
(4) collagen-chitin-polyvinyl alcohol timbering material is immersed in the above-mentioned crosslinker solution 3-5 hour, combines with chondroitin sulfate then and obtains compound collagen stroma tissue engineering support, cleans;
(5) will obtain compound collagen stroma tissue engineering support lyophilization again, and be cut into required size, sterilization packages spare.
More than said cleaning method be to use 0.1M Na
2HPO
4Cleaned compound collagen stroma tissue engineering support two hours, 2MNaCl washed 2 hours, reuse distillation washing.
The compound collagen stroma tissue engineering support that the present invention obtains has good physics, chemical property, and good biocompatibility is easy to operate, and biodegradable absorption; Cell culture experiments shows that cell is well-grown in this support, has no side effect; The pore size of support can be regulated according to clinical needs.The tissue engineering bracket of the present invention's preparation can be used for the structure of artificial organs such as artificial skin, cartilage, blood vessel, and cost is low, the easy easy repetition of method, has potential applicability in clinical practice more widely.
Example 1:
The wet self-conceit pipe cartilage defat of 500g is pulverized, and 37 ℃ of following digestion process 16 hours, adding trichloroacetic acid to final concentration was 15% (w/v) with the buffer solution that contains papain, 0 ℃ of sedimentation 1 hour, and 4 ℃, 8000 rev/mins were removed deproteinize in centrifugal 30 minutes.Add NaOH and NaBH in the supernatant
4Be respectively 0.5M and 0.1M to final concentration.Placed 16 hours for 4 ℃, add 6MHCL and neutralize.Add trichloroacetic acid and the centrifugal deproteinize that removes again.100% ethanol that adds 5 times of volumes in the supernatant ,-20 ℃ of following sedimentations 16 hours, 4 ℃ 8000 rev/mins centrifugal 30 minutes, wash also centrifugal twice of the GAG of gained with 70% ethanol; With dissolved in distilled water and lyophilization.The collagen swelling solution extracts from cattle flesh key by enzymatic isolation method, and its solids content is 1.3%.
Take by weighing collagen swelling solution 50g, it is 0.7% solution that the malonic acid solution with 0.3% is made into concentration as dispersion liquid, stirs the centrifugal bubble of removing.Take by weighing the 0.06g chitosan and be dissolved in the 0.2M acetum, the 0.06g polyvinyl alcohol is dissolved in the 0.3% malonic acid solution, then it is mixed with the collagen swelling solution, and it is also centrifugal to stir.Pour the collagen swelling solution into the lyophilizing container, be lyophilized into spongy thin film with vacuum freeze drier, thickness is about 3mm.This spongy thin film is immersed in 200ml contains in 40% ethanol (pH5.5) of 50mM MES 0.5 hour.Place 200ml to contain 40% alcoholic solution of the 50mM MES of 0.25% (w/v) chondroitin sulfate and 30mM EDC then, react after 4 hours, use 0.1M Na
2HPO
4(pH9.1) cleaned this spongy thin film two hours, 2M NaCl washed 2 hours, and the reuse distillation is washed, and lyophilizing is also sterilized with gamma-radiation.
The compound collagen stroma tissue engineering support porous nickel that the present invention finally obtains, dimensionally stable has certain toughness and intensity.The microcosmic sem photograph (SEM) of the compound collagen stroma tissue engineering support that from Fig. 1, provides as can be seen, this support has the continuous three-dimensional fibrillar meshwork structure, the aperture is about 30-100 μ m.Adopt photoelectron spectroscopy PHI5300 ESCA system that the composition of material surface is analyzed as can be known, the adding of chitosan can make the binding capacity of chondroitin sulfate obviously increase (seeing Table 1); This compound collagen stroma tissue engineering support material has good biocompatibility, the L929 cell is after cultivating 7 days on the support, the cell growth in flakes, Fig. 2, the 3rd, the microcosmic sem photograph (SEM) of compound collagen stroma tissue engineering support, provided the cell combination among the figure respectively and not in conjunction with the SEM figure of growth in the compound collagen stroma tissue engineering support of chondroitin sulfate after 7 days, the result shows that the compound collagen stroma tissue engineering support that combines chondroitin sulfate has played the effect that promotes cell proliferation.
Example 2:
The extraction of chondroitin sulfate and collagen swelling solution such as example 1.Take by weighing collagen swelling solution 50g, the malonic acid solution with 0.3% is as dispersion liquid, is made into concentration and is 0.6% solution, stirs the centrifugal bubble of removing.Take by weighing the 0.08g chitosan and be dissolved in the 0.2M acetum, the 0.08g polyvinyl alcohol is dissolved in the 0.3% malonic acid solution, and it is mixed with the collagen swelling solution, and it is also centrifugal to stir.This swelling solution is poured in the lyophilizing container, be lyophilized into the compound collagen stroma tissue engineering support of thick about 3mm with vacuum freeze drier, support is immersed in 200ml contains in 40% ethanol (pH5.5) of 50mM MES 0.5 hour, place 200ml to contain 40% alcoholic solution of MES of the 50mM of 0.20% (w/v) chondroitin sulfate and 30mMEDC then, react after 4 hours, use 0.1MNa
2HPO
4(pH9.1) cleaned 2 hours, 2M NaCl washed 2 hours, and reuse distillation washing and lyophilizing obtain product.
Example 3:
The extraction of chondroitin sulfate and collagen swelling solution such as example 1.Take by weighing collagen swelling solution 50g, the malonic acid solution with 0.3% is as dispersion liquid, is made into concentration and is 0.8% solution, stirs the centrifugal bubble of removing.Take by weighing the 0.05g chitosan and be dissolved in the 0.2M acetum, the 0.06g polyvinyl alcohol is dissolved in the 0.3% malonic acid solution, and it is mixed with the collagen swelling solution, and it is also centrifugal to stir.Pour this solution into the lyophilizing container, be lyophilized into the compound collagen stroma tissue engineering support of thick about 3mm with vacuum freeze drier, this support is immersed in 200ml contains in 40% ethanol (pH5.5) of 50mM MES 0.5 hour, place 200ml to contain 40% alcoholic solution of the 50mM MES of 0.3% (w/v) chondroitin sulfate and 30mMEDC then, react after 4 hours, use 0.1M Na
2HPO
4(pH9.1) cleaned substrate 2 hours, 2M NaCL washed 2 hours, and reuse distillation washing and lyophilizing obtain product.
Table 1 esca analysis stromal surface is elementary composition
| ????C | ????N | ????O | ????S | |
| Collagen-CS collagen-CS-chitosan blank | ??67.46％ ??62.71％ ??74.80％ | ??19.52％ ??23.28％ ??15.78％ | ????12.02％ ????12.57％ ????9.25％ | ????1.00％ ????1.43％ ????0.17％ |
Claims (4)
1, a kind of compound collagen stroma tissue engineering support is characterized in that its weight composition is: 10 parts of collagens, chondroitin sulfate 1-5 part, chitosan 0.5-3 part, polyvinyl alcohol 0.5-2 part.
2, the preparation method of the said compound collagen stroma tissue engineering support of claim 1 is characterized in that it comprises the steps:
(1) be the collagen malonic acid swelling solution of 0.4-1.0% by metering configuration solids content, acetum dissolving chitosan with 0.2M, 0.3% malonic acid solution dissolve polyvinyl alcohol, and with in its adding collagen swelling solution, stirring obtains collagen-chitin-poly-vinyl alcohol solution;
(2) the centrifugal bubble of removing places the lyophilizing container, and control cryogenic temperature-30 ℃ obtains porous collagen-chitin-polyvinyl alcohol timbering material to-10 ℃;
(3) from self-conceit pipe cartilage, extract chondroitin sulfate with the papain digestion method; Concentration is dissolved in the crosslinker solution for the 0.1-0.5% chondroitin sulfate, and crosslinker solution is the 20-40% alcoholic solution that contains the 2N-morphine quinoline-ethyl sulfonic acid (MES) of 1-ethyl-3-3-dimethylaminopropyl-carbonization two imido (EDC);
(4) collagen-chitin-polyvinyl alcohol timbering material is immersed in the above-mentioned crosslinker solution 3-5 hour, combines with chondroitin sulfate and obtains compound collagen stroma tissue engineering support, cleans then;
(5) will obtain compound collagen stroma tissue engineering support lyophilization again, and be cut into required size, sterilization packages spare.
3,, it is characterized in that said cleaning method is to use 0.1M Na according to the preparation method of the said compound collagen stroma tissue engineering support of claim 2
2HPO
4Cleaned compound collagen stroma tissue engineering support two hours, 2M NaCl washed 2 hours, reuse distillation washing.
4,, it is characterized in that said sterilizing methods is to sterilize with gamma-radiation according to the preparation method of the said compound collagen stroma tissue engineering support of claim 2.
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| CNB01109205XA CN1141150C (en) | 2001-02-28 | 2001-02-28 | Compound collagen stroma tissue engineering support and preparation method thereof |
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| CNB01109205XA CN1141150C (en) | 2001-02-28 | 2001-02-28 | Compound collagen stroma tissue engineering support and preparation method thereof |
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Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1319605C (en) * | 2005-12-07 | 2007-06-06 | 浙江大学 | Collagen-chitin and silicon rubber bilayer skin regeneration support and its preparation method |
| WO2007124622A1 (en) * | 2006-04-28 | 2007-11-08 | Wuhan University Of Technology | The 3d porous layered scaffold for tissue engineering and the preparation thereof |
| CN100372576C (en) * | 2004-12-29 | 2008-03-05 | 东华大学 | Composite collagen nerve ductus for promoting neural regeneration, and method for forming filature from hollow wet process |
| CN101032430B (en) * | 2007-04-13 | 2011-03-16 | 中国人民解放军第三军医大学第一附属医院 | Method for preparing integrated frame of cartilage of tissue-engineered bone having function interface |
| CN101690830B (en) * | 2009-09-29 | 2012-12-05 | 中国人民解放军第三军医大学第一附属医院 | Preparation method of bionic cartilage extracellular matrix for tissue engineering |
| CN103083722A (en) * | 2011-11-08 | 2013-05-08 | Hoya株式会社 | Artificial cartilage and its production method |
| CN104211982A (en) * | 2013-05-31 | 2014-12-17 | 嘉兴学院 | Manufacturing method of multi-aperture tissue engineering scaffold for cell growth |
| CN105963779A (en) * | 2016-07-01 | 2016-09-28 | 赵艳丽 | Bone repairing material and preparation method thereof |
| CN106693050A (en) * | 2017-02-28 | 2017-05-24 | 四川大学 | Preparation method for composite scaffold material based on collagen and collagen fibers |
| CN106823012A (en) * | 2017-02-09 | 2017-06-13 | 沈坤 | Medical material with bacteria resistance function and preparation method thereof, application |
| CN106924809A (en) * | 2017-03-20 | 2017-07-07 | 北京华信佳音医疗科技发展有限责任公司 | Filler under a kind of I-type collagen and liquid mucous membrane |
| CN108888384A (en) * | 2018-07-18 | 2018-11-27 | 广州迈普再生医学科技股份有限公司 | A kind of tubular bracket and preparation method thereof with double-layer structure |
| CN110452413B (en) * | 2019-07-08 | 2021-05-18 | 南京中富先农生物科技有限公司 | Collagen cross-linking agent composition and application thereof |
-
2001
- 2001-02-28 CN CNB01109205XA patent/CN1141150C/en not_active Expired - Fee Related
Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100372576C (en) * | 2004-12-29 | 2008-03-05 | 东华大学 | Composite collagen nerve ductus for promoting neural regeneration, and method for forming filature from hollow wet process |
| CN1319605C (en) * | 2005-12-07 | 2007-06-06 | 浙江大学 | Collagen-chitin and silicon rubber bilayer skin regeneration support and its preparation method |
| WO2007124622A1 (en) * | 2006-04-28 | 2007-11-08 | Wuhan University Of Technology | The 3d porous layered scaffold for tissue engineering and the preparation thereof |
| CN100453124C (en) * | 2006-04-28 | 2009-01-21 | 武汉理工大学 | Porous, laminated, tri-dimensional multiple-grade structure tissue stent material and its preparation method |
| CN101032430B (en) * | 2007-04-13 | 2011-03-16 | 中国人民解放军第三军医大学第一附属医院 | Method for preparing integrated frame of cartilage of tissue-engineered bone having function interface |
| CN101690830B (en) * | 2009-09-29 | 2012-12-05 | 中国人民解放军第三军医大学第一附属医院 | Preparation method of bionic cartilage extracellular matrix for tissue engineering |
| CN103083722A (en) * | 2011-11-08 | 2013-05-08 | Hoya株式会社 | Artificial cartilage and its production method |
| CN104211982A (en) * | 2013-05-31 | 2014-12-17 | 嘉兴学院 | Manufacturing method of multi-aperture tissue engineering scaffold for cell growth |
| CN105963779A (en) * | 2016-07-01 | 2016-09-28 | 赵艳丽 | Bone repairing material and preparation method thereof |
| CN106823012A (en) * | 2017-02-09 | 2017-06-13 | 沈坤 | Medical material with bacteria resistance function and preparation method thereof, application |
| CN106693050A (en) * | 2017-02-28 | 2017-05-24 | 四川大学 | Preparation method for composite scaffold material based on collagen and collagen fibers |
| CN106693050B (en) * | 2017-02-28 | 2019-09-13 | 四川大学 | A kind of preparation method of the compound support frame material based on collagen and collagenous fibres |
| CN106924809A (en) * | 2017-03-20 | 2017-07-07 | 北京华信佳音医疗科技发展有限责任公司 | Filler under a kind of I-type collagen and liquid mucous membrane |
| CN108888384A (en) * | 2018-07-18 | 2018-11-27 | 广州迈普再生医学科技股份有限公司 | A kind of tubular bracket and preparation method thereof with double-layer structure |
| CN110452413B (en) * | 2019-07-08 | 2021-05-18 | 南京中富先农生物科技有限公司 | Collagen cross-linking agent composition and application thereof |
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|---|---|
| CN1141150C (en) | 2004-03-10 |
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