CN103739869B - The preparation method of a kind of cross-linked chitosan/hyaluronic acid complex stephanoporate bracket - Google Patents
The preparation method of a kind of cross-linked chitosan/hyaluronic acid complex stephanoporate bracket Download PDFInfo
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- CN103739869B CN103739869B CN201310724892.5A CN201310724892A CN103739869B CN 103739869 B CN103739869 B CN 103739869B CN 201310724892 A CN201310724892 A CN 201310724892A CN 103739869 B CN103739869 B CN 103739869B
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- hyaluronic acid
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- KVZPGSHMEVUWNJ-UHFFFAOYSA-N CO[Si](CCCOCC1ON1)(OC)OC Chemical compound CO[Si](CCCOCC1ON1)(OC)OC KVZPGSHMEVUWNJ-UHFFFAOYSA-N 0.000 description 1
Abstract
The invention belongs to new material technology field, be specifically related to the preparation method of a kind of cross-linked chitosan/hyaluronic acid complex stephanoporate bracket.The method is for raw material with the natural biologic material such as chitosan and hyaluronic acid, organo silane coupling agent KH-560 is linking agent, adopt freeze-drying, prepare the cross-linked chitosan be made up of the pure-natural biological material/hyaluronic acid complex stephanoporate bracket of good mechanical performance.Cross-linked chitosan prepared by the present invention/hyaluronic acid complex stephanoporate bracket has the features such as high porosity, high hole strength and wide aperture, its aperture is 100 ~ 300 μm, porosity > 90%, for macropore is containing the opening pore structure of aperture, is adapted at rack surface repopulating cell.Compare chitosan stent, this cross-linked chitosan/hyaluronic acid complex stephanoporate bracket has better mechanical property, adding of hyaluronic acid and organosilicon cross-linking agent, is more conducive to cell in porous scaffold surface propagation and differentiation, better to the affinity of biomass cells.
Description
Technical field
The invention belongs to new material technology field, be specifically related to the preparation method of a kind of cross-linked chitosan/hyaluronic acid complex stephanoporate bracket.
Background technology
Appear as human health and the life of organizational project extend and bring fine prospect, utilize biomedical material to prepare artificial substituent and carry out disease therapy, can solve human organ because of defect or afunction need to change, limited difficult problem of drawing materials.When people invest artificial tissue or organ energy, one of its key is the preparation of the organizational project three-dimensional porous rack material utilizing cell cultures.
At present, tissue engineered porous scaffold material is mainly from some natural polymers and synthesis polymer.Natural polymer mainly contains collagen, chitin, chitosan, hyaluronic acid, gelatin, alginates, chondroitin sulfate etc.; Synthesis polymer mainly contains poly(lactic acid), polyglycolic acid, polyhydroxybutyrate, polycaprolactone, poe etc.Nature is used in line with facilitating, being easy to get, take from nature, utilize the close and similar advantage held altogether of natural biologic material and tissue, many investigators have selected natural biologic material and prepare tissue engineering bracket material, but natural biologic material ubiquity the weak problem of physical strength, this selects suitable natural biologic material to carry out composite with regard to needing people, and select effective linking agent to improve its mechanical property, this linking agent must be also no cytotoxicity.
Secondly, as effective tissue engineering bracket, light has material itself to be inadequate, also biomaterial will be made the three-dimensional porous rack with specified shape and pore structure.In order to cell can enter support, require that support has vesicular structure, and there is a certain size aperture and the opening structure of hole strength, and the aperture of different directions is identical.Therefore, the preparation method of tissue engineered porous scaffold and technology also most important.Its pore method mainly contains particle stripping method, phase separation method/freeze-drying, gas foaming method, sintering microballoon method etc.But how to select suitable pore method, prepare the organizational project three-dimensional porous rack met certain requirements, rack surface is made to have cellular affinity, and surface micro-structure communicates as wide aperture (100 ~ 300 μm), high porosity (>90%), hole, hole and certain hole strength etc. is conducive to the adhesion of cell, propagation and differential growth, remains a technical barrier.
Summary of the invention
The object of the invention is to the deficiency overcoming prior art existence, the preparation method of a kind of cross-linked chitosan/hyaluronic acid complex stephanoporate bracket is provided.
The preparation method of a kind of cross-linked chitosan/hyaluronic acid complex stephanoporate bracket that the present invention proposes, the method is for raw material with the natural biologic material such as chitosan and hyaluronic acid, organo silane coupling agent KH-560 is linking agent, adopt freeze-drying, prepare the cross-linked chitosan be made up of the pure-natural biological material/hyaluronic acid complex stephanoporate bracket of good mechanical performance.Cross-linked chitosan prepared by the present invention/hyaluronic acid complex stephanoporate bracket has the features such as high porosity, high hole strength and wide aperture, its aperture is 100 ~ 300 μm, porosity > 90%, for macropore is containing the opening pore structure of aperture, is adapted at rack surface repopulating cell.
The preparation method of a kind of cross-linked chitosan/hyaluronic acid complex stephanoporate bracket that the present invention proposes, concrete steps are as follows:
(1) configuration quality per-cent is the methanol solution of the methanol suspension of the chitosan of 1 ~ 10%, hyaluronic methanol suspension and silane coupling agent KH-560 respectively;
(2) methanol solution 20 ~ 40:20 in mass ratio ~ 40:20 ~ 40 of the methanol suspension of chitosan step (1) obtained, hyaluronic methanol suspension and silane coupling agent KH-560 are blended, wherein, the mol ratio of chitosan, hyaluronic acid and silane coupling agent KH-560 is 1 ~ 2:1 ~ 2:2 ~ 4, be warming up to 60 ~ 90 DEG C, stirring reaction 1 ~ 6 hour, suction filtration, repeatedly wash with anhydrous methanol, until wash not reacted silane coupling agent KH-560,50 ~ 70 DEG C of vacuum-dryings, after 6 ~ 24 hours, obtain cross-linked chitosan/hyaluronic acid composite;
(3) by cross-linked chitosan/hyaluronic acid composite that step (2) obtains, be dissolved in 50 ~ 250g distilled water, join mass percent is the aqueous solution of the cross-linked chitosan/hyaluronic acid composite of 1 ~ 10%, deaeration, obtains the aqueous solution of the cross-linked chitosan/hyaluronic acid composite after deaeration;
(4) aqueous solution of the cross-linked chitosan/hyaluronic acid composite after deaeration step (3) obtained, pour in micro-container of 10 mL, wait prolonging, levelling is whole to be placed in-120 ~-20 DEG C of refrigeration chambers after freezing 12 ~ 48 hours, to move in freeze drier lyophilize again 6 ~ 36 hours, namely obtain a kind of cross-linked chitosan/hyaluronic acid complex stephanoporate bracket.
In the present invention, described chitosan is β-Isosorbide-5-Nitrae-poly-glucosamine, and its chemical structural formula is as follows:
。
In the present invention, the higher polysaccharides class that described hyaluronic acid is made up of dissacharide units D-Glucose aldehydic acid and N-acetyl-glucosamine, its chemical structural formula is as follows:
。
In the present invention, described silane coupling agent KH-560 is γ-(2,3-epoxy third oxygen) propyl trimethoxy silicane, and its chemical structural formula is as follows:
。
Compared with prior art, advantage of the present invention is: 1. the present invention is the preparation method of a kind of cross-linked chitosan/hyaluronic acid complex stephanoporate bracket, for raw material with the natural biologic material such as chitosan and hyaluronic acid, with the organo silane coupling agent KH-560 of good biocompatibility for linking agent, adopt freeze-drying, the cross-linked chitosan/hyaluronic acid complex stephanoporate bracket of obtained good mechanical performance; 2. cross-linked chitosan/hyaluronic acid complex stephanoporate bracket that prepared by the present invention has aperture large (100 ~ 300 μm), porosity high (> 90%), and macropore, containing the opening pore structure of aperture, is adapted at rack surface repopulating cell; 3. this cross-linked chitosan/hyaluronic acid complex stephanoporate bracket has better mechanical property, adding of organosilicon cross-linking agent, not only there is not cytotoxicity, be conducive to cell all the better at the propagation of porous scaffold surface and differential growth, good to the affinity of biomass cells; 4. preparation method of the present invention has the advantages such as technique is simple and convenient, quality product is high, application prospect is extensive, the product utilizing the present invention to prepare is high-performance organizational project three-dimensional porous rack of new generation, the pore morphology that there is porous, high porosity and be connected, possess suitable mechanical strength, be conducive to the advantages such as the adhesion of cell, propagation and differentiation.
Accompanying drawing explanation
Fig. 1 is the preparation flow figure of cross-linked chitosan/hyaluronic acid complex stephanoporate bracket.
Fig. 2 is the scanning electron microscope (SEM) photograph of cross-linked chitosan/hyaluronic acid complex stephanoporate bracket.Wherein: (a) magnification is 100, (b) magnification is 400.
Embodiment
The present invention is further illustrated below by embodiment.
Embodiment 1
Configuration quality per-cent is the methanol solution of the methanol suspension of the chitosan of 2%, hyaluronic methanol suspension and silane coupling agent KH-560 respectively;
Be that 35:35:30 is blended in mass ratio by the methanol solution of the methanol suspension of above-mentioned chitosan, hyaluronic methanol suspension and silane coupling agent KH-560, be warming up to 70 DEG C, stirring reaction 3 hours, suction filtration, repeatedly wash with anhydrous methanol, until wash not reacted silane coupling agent KH-560,60 DEG C of vacuum-dryings, after 24 hours, obtain cross-linked chitosan/hyaluronic acid composite;
By above-mentioned cross-linked chitosan/hyaluronic acid composite, be dissolved in 100g distilled water, join mass percent is the aqueous solution of the cross-linked chitosan/hyaluronic acid composite of 1%, deaeration, obtains the aqueous solution of the cross-linked chitosan/hyaluronic acid composite after deaeration;
By the aqueous solution of the cross-linked chitosan/hyaluronic acid composite after above-mentioned deaeration, pour in micro-container of 10 mL, wait prolonging, levelling is whole to be placed in-30 DEG C of refrigeration chambers after freezing 12 hours, to move in freeze drier lyophilize again 24 hours, namely obtain a kind of cross-linked chitosan/hyaluronic acid complex stephanoporate bracket.
The scanning electron microscope (SEM) photograph of Fig. 1 to be the preparation flow figure of cross-linked chitosan/hyaluronic acid complex stephanoporate bracket, Fig. 2 be cross-linked chitosan/hyaluronic acid complex stephanoporate bracket.
Embodiment 2
Identical with embodiment 1, but the concentration of the methanol solution of the methanol suspension of the middle chitosan of step (1), hyaluronic methanol suspension and silane coupling agent KH-560 becomes 3% by 2%, and other are constant.
Embodiment 3
Identical with embodiment 1, but the concentration of the methanol solution of the methanol suspension of the middle chitosan of step (1), hyaluronic methanol suspension and silane coupling agent KH-560 becomes 1% by 2%, and other are constant.
Embodiment 4
Identical with embodiment 1, but the mass ratio of the methanol solution of the methanol suspension of the middle chitosan of step (2), hyaluronic methanol suspension and silane coupling agent KH-560 becomes 30:30:40 from 35:35:30, and other are constant.
Embodiment 5
Identical with embodiment 1, but the mass ratio of the methanol solution of the methanol suspension of the middle chitosan of step (2), hyaluronic methanol suspension and silane coupling agent KH-560 becomes 40:40:20 from 35:35:30, and other are constant.
Embodiment 6
Identical with embodiment 1, but in step (3), the mass percent of the aqueous solution of cross-linked chitosan/hyaluronic acid composite becomes 2% from 1%, and other are constant.
Embodiment 7
Identical with embodiment 1, but in step (3), the mass percent of the aqueous solution of cross-linked chitosan/hyaluronic acid composite becomes 3% from 1%, and other are constant.
Embodiment 8
Identical with embodiment 1, but in step (3), the mass percent of the aqueous solution of cross-linked chitosan/hyaluronic acid composite becomes 5% from 1%, and other are constant.
Embodiment 9
Identical with embodiment 1, but in the middle refrigeration chamber of step (4), freezing temp becomes-50 DEG C from-30 DEG C, and other are constant.
Embodiment 10
Identical with embodiment 1, but in the middle refrigeration chamber of step (4), freezing temp becomes-100 DEG C from-30 DEG C, and other are constant.
The product of the cross-linked chitosan obtained in embodiment 2-10/hyaluronic acid complex stephanoporate bracket and embodiment 1 has similar performance.
Claims (3)
1. a preparation method for cross-linked chitosan/hyaluronic acid complex stephanoporate bracket, is characterized in that concrete steps are as follows:
(1) configuration quality per-cent is the methanol solution of the methanol suspension of the chitosan of 1 ~ 10%, hyaluronic methanol suspension and silane coupling agent KH-560 respectively;
(2) methanol solution 20 ~ 40:20 in mass ratio ~ 40:20 ~ 40 of the methanol suspension of chitosan step (1) obtained, hyaluronic methanol suspension and silane coupling agent KH-560 are blended, wherein, the mol ratio of chitosan, hyaluronic acid and silane coupling agent KH-560 is 1 ~ 2:1 ~ 2:2 ~ 4, be warming up to 60 ~ 90 DEG C, stirring reaction 1 ~ 6 hour, suction filtration, repeatedly wash with anhydrous methanol, until wash not reacted silane coupling agent KH-560,50 ~ 70 DEG C of vacuum-dryings, after 6 ~ 24 hours, obtain cross-linked chitosan/hyaluronic acid composite;
(3) by cross-linked chitosan/hyaluronic acid composite that step (2) obtains, be dissolved in 50 ~ 250g distilled water, join mass percent is the aqueous solution of the cross-linked chitosan/hyaluronic acid composite of 1 ~ 10%, deaeration, obtains the aqueous solution of the cross-linked chitosan/hyaluronic acid composite after deaeration;
(4) aqueous solution of the cross-linked chitosan/hyaluronic acid composite after deaeration step (3) obtained, pour in micro-container of 10 mL, wait prolonging, levelling is whole to be placed in-120 ~-20 DEG C of refrigeration chambers after freezing 12 ~ 48 hours, to move in freeze drier lyophilize again 6 ~ 36 hours, namely obtain a kind of cross-linked chitosan/hyaluronic acid complex stephanoporate bracket.
2. the preparation method of a kind of cross-linked chitosan/hyaluronic acid complex stephanoporate bracket according to claim 1, is characterized in that, described chitosan is β-Isosorbide-5-Nitrae-poly-glucosamine, and its chemical structural formula is as follows:
。
3. the preparation method of a kind of cross-linked chitosan/hyaluronic acid complex stephanoporate bracket according to claim 1, it is characterized in that, the higher polysaccharides class that described hyaluronic acid is made up of dissacharide units D-Glucose aldehydic acid and N-acetyl-glucosamine, its chemical structural formula is as follows:
。
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1342722A (en) * | 2001-09-26 | 2002-04-03 | 天津大学 | Process for preparing dual-layer combined chitosan-gelatin-mucinase scaffold material |
| CN1613514A (en) * | 2004-11-21 | 2005-05-11 | 厦门大学 | Three-dimensional porous tissue engineering stand material and preparation thereof |
| WO2007111416A1 (en) * | 2006-03-28 | 2007-10-04 | Korea Atomic Energy Research Institute | Method of producing chitosan scaffold having high tensile strength and chitosan scaffold produced using the method |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1342722A (en) * | 2001-09-26 | 2002-04-03 | 天津大学 | Process for preparing dual-layer combined chitosan-gelatin-mucinase scaffold material |
| CN1613514A (en) * | 2004-11-21 | 2005-05-11 | 厦门大学 | Three-dimensional porous tissue engineering stand material and preparation thereof |
| WO2007111416A1 (en) * | 2006-03-28 | 2007-10-04 | Korea Atomic Energy Research Institute | Method of producing chitosan scaffold having high tensile strength and chitosan scaffold produced using the method |
Non-Patent Citations (1)
| Title |
|---|
| Comparative analysis of gelatin scaffolds crosslinked by genipin and silane coupling agent;C.Tonda-Turo等;《International Journal of Biological Macromolecules》;20110713;700-706 * |
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