CN1493366A - Preparation method of cell substrate protein biological support material used for tissue engineering - Google Patents
Preparation method of cell substrate protein biological support material used for tissue engineering Download PDFInfo
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- CN1493366A CN1493366A CNA031345379A CN03134537A CN1493366A CN 1493366 A CN1493366 A CN 1493366A CN A031345379 A CNA031345379 A CN A031345379A CN 03134537 A CN03134537 A CN 03134537A CN 1493366 A CN1493366 A CN 1493366A
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- stromatin
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Abstract
A cytostromatic protein bioscaffold material for tissue engineering is prepared through using the buffering acetic acid solution to extract the cytostromatic protein, depositing with inorganic salt, and using the inorganic salt with different gradient to remove the III-type collagen from the cytostromatic protein, resulting in purified cytostromatic protein. Its advantages are high stability, purity and adhesion and no toxic by-effect.
Description
Technical field
The invention belongs to the biomaterial preparing technical field, relate to a kind of cellular matrix protein biology preparation methods that is used for organizational project.
Background technology
Stromatin is the major protein composition of connective tissue, has good mechanical performance as the timbering material of organizational project, and its major reason is that stromatin can be natural crosslinked, the three dimensional structure that the formative tissue engineering product is required.Therefore, stromatin can satisfy the requirement of organizational project biomaterial in scope widely.At present, stromatin composition (as collagen, chondroitin sulfate, extracellular matrix) is widely used aspect medicine and pharmacology, as can be used for the agent of surgery burn treating, as pharmaceutical carrier or repair materials (ointment, suture) etc.But the application aspect organizational project is had relatively high expectations to stromatin, should as much as possible it be treated to form solubility in acid, non-degeneration, non-allergic effect, no bad impurity.Disclosed patent application in 1994 " extraction of human placental collagen and preparation method thereof ", this method utilize people's Placenta Hominis through clean, smash, extraction, mixing form.There is following shortcoming in it: 1. composition is single, only is the collagen composition; Material behaviour Placenta Hominis, it is limited to originate, and is not suitable as the extensive use of organizational project; 2. the final products of Huo Deing are liquid, can't detect product content; 3. product does not have plasticity, can't be used for the development of tissue engineering product.And disclosed patent application in 1999 " medical collagen sponge and manufacturing process thereof " final products also only are the collagen composition.Its shortcoming is: 1. material use Protease Treatment, destroyed proteic native configurations; 2. material is carried out crosslinkedly, destroyed proteic biocompatibility.
Summary of the invention
At above-mentioned defective of the prior art, the preparation method that the purpose of this invention is to provide a kind of cellular matrix protein biology timbering material of the adjustable degradability with solubility in acid, non-degeneration and non-allergic effect, prepared material can obviously improve the adhesiveness of seed cell to material, and the biocompatibility of material.
Because stromatin has low immunogenicity, compares with other protein with immunity, has superior biocompatibility and safety, is considered to one of the most useful biomaterial.Therefore, the cellular matrix protein biology material that is used for organizational project that the present invention is prepared is purification and albumen non-degeneration, based on I, IV collagen protein, and contains hyaluronic acid, proteoglycan and the multiple cytokine that helps cell proliferation.To the biomaterial performance demands, preparation method of the present invention comprises cleaning, smashes, the step of extraction, mixing, purification according to these, and its concrete operations step is:
1. will be broken for granule after tire cattle corium, tendon, the osseous tissue washing;
2. with Tris/edta solution washing tissue particles;
3. from tissue particles, extract stromatin with 0.1～0.5M acetate buffer;
4. in extracting solution, add inorganic salt, the rough stromatin of salt precipitation;
5. will precipitate with 0.1～0.5M acetate buffer and dissolve;
6. reuse inorganic salt salt precipitation obtains the stromatin crude product;
7. with TRIS buffer (pH=7.0) dissolved matrix albumen crude product;
8. remove the III Collagen Type VI with different inorganic salt concentration gradients, obtain the stromatin of purification;
9. with 0.05～0.2M acetate buffer dissolving purification stromatin;
10. with sodium hydroxide solution this stromatin solution of dialysing, be neutral;
11. distill water dialysis 3～6 times;
12. obtain the stromatin product with freeze drying process, it is the milky spongiosis;
13. after the product sterilization, 4 ℃ of preservations.
In said method, both can remove in the raw material and irrelevant impurity of cellular matrix albumen such as fat etc., the dissolving of stromatin capable of inhibiting cell again with Tris/edta solution.Being neutral reason by alkaline solution dialysis stromatin solution is because the product that prior art obtains is generally faintly acid, can't be directly and cell compound, must carry out aquation during application, influence seed cell to the adhering to of material, thereby influenced the application of product in organizational project.
The cellular matrix albumen height of collagen content in the said method in the cellular matrix albumen crude product after than purification, it can be used for the zoopery of Tissue Engineering Study, and the proteic immunogenicity of the cellular matrix behind the purification is lower, human body is had no side effect, can extract the research and development that are used for tissue engineering product in a large number.
The cellular matrix albumen of the present invention's preparation has biodegradable and reproducibility, in vivo can be by most protease hydrolysiss, and Here it is, and stromatin has the principle of biodegradable.Prepared stromatin has the formation again of fiber simultaneously, and purified dissolvable matrix albumen can be in the external formation once more ordered fiber shape structure similar to natural collagen fibre.The stromatin of the present invention's preparation has excellent biological compatibility, no matter the stromatin material is to be absorbed previous crops for forming neoblastic skeleton, still be absorbed assimilation and enter the host, become the part of host tissue, all good interaction is arranged with pericellular substrate, show excellent biological compatibility, can become a part gradually with normal physiological function cell and organized whole.Simultaneously, also can promote the growth of dissimilar cells.
Do not use cross-linking agent glutaraldehyde or resorcinol in the preparation method of the present invention, enlarged because of toxicity limits its scope of application, cellular matrix albumen can be absorbed by body behind certain hour.Also can utilize it as the low molding medical material of immunity with good strength.Cellular matrix albumen of the present invention is that sponge is membranaceous, can be directly used in the structure of tissue engineering product, also make type pipe, sheet material, net etc. with the method for injecting after the solubilized, simultaneously can be with other artificial or natural material be combined to form organizational project and the dimensional culture that gel is used for skin, blood vessel, dental pulp.
Compared with prior art, the invention has the advantages that: prepared cellular matrix protein biology material is non-degeneration, purity height, can be prepared as different shape according to different application requirements, as gel, spongy, membranaceous, glue sheet or liquid; Can be widely used in organizational project and dimensional culture.Kept proteic biologic activity, pair cell stick and propagation has facilitation.This material has good mechanical performance, degradation speed is adjustable, good biocompatibility, body is had no side effect; And can be compound with other biological material (as chitosan, polylactic acid/polyglycolic acid, calcium phosphate etc.), be made into organizational project and dimensional culture that mandruka, gel, film, microgranule etc. are widely used in skin, bone, cartilage, dental pulp, periodontal as required.Compare with the high molecular polymer that is used for the organizational project biomaterial at present, can obviously improve the adhesion characteristics of seed cell, reduce the cytotoxicity of material and improve the preparation technology of timbering material material.In addition, raw material sources of the present invention are extensive, and are cheap, but mass preparation.
The specific embodiment
Now the inventive method is described further by following embodiment
Embodiment 1
1) will be broken for granule after tire cattle corium, tendon, the osseous tissue washing;
2) washed tissue particles 24 hours, changed liquid 1 time in per 6 hours with 0.1M Tris/1: 1 mixed liquor of 1mM ethylenediaminetetraacetic acid;
3) from tissue particles, extract stromatin 72 hours with the 0.5M acetate buffer, changed liquid 1 time in per 24 hours;
4) extracting solution is passed through to add 1M NaCl salt precipitation;
5) reuse 0.5M acetate buffer dissolution precipitation;
6) add 1M NaCl salt precipitation again, obtain the stromatin crude product;
7) use distill water dialysis 4 times, remove residual acetate buffer;
8) obtain the stromatin crude product after the lyophilization, the back 4 ℃ of preservations of sterilizing.
Embodiment 2
With among the embodiment 1 6) the stromatin crude product that obtains is again by following processing:
1) dissolves rough albumen with 0.1M TRIS buffer (pH 7.0);
2) filter and purification solution with 200 mesh filter screens;
3) in solution, add different gradient NaCl and remove the III Collagen Type VI, obtain the stromatin of purification;
4) use distill water dialysis 5 times, remove residual acetate buffer;
5) obtain the pure product of stromatin (meta-acid) after the lyophilization, the back 4 ℃ of preservations of sterilizing.
Embodiment 3
With among the embodiment 2 3) the pure product of stromatin that obtain are again by following processing:
1) with the stromatin behind the 0.05M acetate buffer dissolving purification;
2), be neutral by sodium hydroxide solution this stromatin solution of dialysing;
3) distill water dialysis is 6 times, removes the residual acetic acid buffer;
4)-30 a ℃ lyophilization obtains the stromatin product, and it is the milky spongiosis;
5) after the product sterilization, 4 ℃ of preservations.
Claims (3)
1. a cellular matrix protein biology preparation methods that is used for organizational project comprises cleaning, smashes, extracts.Purification step is characterized in that, operating procedure is:
(1) will be broken for granule after tire cattle corium, tendon, the osseous tissue washing;
(2) with Tris/edta solution washing tissue particles;
(3) from tissue particles, extract stromatin with the 0.1-0.5M acetate buffer;
(4) in extracting solution, add inorganic salt, salt precipitation;
(5) will precipitate with the dissolving of 0.1-0.5M acetate buffer;
(6) reuse inorganic salt salt precipitation obtains cellular matrix albumen crude product;
(7) distill water dialysis is 3-6 time;
(8) obtain the cellular matrix protein product with freeze drying process, after the sterilization, 4 ℃ of preservations.
2, preparation method according to claim 1 is characterized in that, also has between described step (6) and (7):
(1) with TRIS buffer dissolved matrix albumen crude product;
(2) remove the III Collagen Type VI with different inorganic salt concentration gradients, obtain the stromatin of purification.
3, preparation method according to claim 2 is characterized in that, the purifying cells stromatin that obtains is done following processing:
(1) with the stromatin behind the 0.05-0.2M acetate buffer dissolving purification;
(2), be neutral by sodium hydroxide solution this stromatin solution of dialysing.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA031345379A CN1493366A (en) | 2003-09-02 | 2003-09-02 | Preparation method of cell substrate protein biological support material used for tissue engineering |
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| Application Number | Priority Date | Filing Date | Title |
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| CNA031345379A CN1493366A (en) | 2003-09-02 | 2003-09-02 | Preparation method of cell substrate protein biological support material used for tissue engineering |
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| CN1493366A true CN1493366A (en) | 2004-05-05 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100423795C (en) * | 2005-11-23 | 2008-10-08 | 曲彦隆 | Derivative tendon stent material and its preparing process |
| CN101234215B (en) * | 2008-03-06 | 2010-12-01 | 西安组织工程工程技术研究中心 | Cell-less composite type artificial skin and preparation thereof |
-
2003
- 2003-09-02 CN CNA031345379A patent/CN1493366A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100423795C (en) * | 2005-11-23 | 2008-10-08 | 曲彦隆 | Derivative tendon stent material and its preparing process |
| CN101234215B (en) * | 2008-03-06 | 2010-12-01 | 西安组织工程工程技术研究中心 | Cell-less composite type artificial skin and preparation thereof |
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