CN108310462A - A kind of preparation method of hydroxyethyl chitosan plural gel - Google Patents
A kind of preparation method of hydroxyethyl chitosan plural gel Download PDFInfo
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- CN108310462A CN108310462A CN201810378975.6A CN201810378975A CN108310462A CN 108310462 A CN108310462 A CN 108310462A CN 201810378975 A CN201810378975 A CN 201810378975A CN 108310462 A CN108310462 A CN 108310462A
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/26—Mixtures of macromolecular compounds
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/52—Hydrogels or hydrocolloids
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- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
- C08B37/0027—2-Acetamido-2-deoxy-beta-glucans; Derivatives thereof
- C08B37/003—Chitin, i.e. 2-acetamido-2-deoxy-(beta-1,4)-D-glucan or N-acetyl-beta-1,4-D-glucosamine; Chitosan, i.e. deacetylated product of chitin or (beta-1,4)-D-glucosamine; Derivatives thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/06—Materials or treatment for tissue regeneration for cartilage reconstruction, e.g. meniscus
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Abstract
The present invention relates to a kind of preparation methods of hydroxyethyl chitosan plural gel, use special process to carry out hydroxyethylation to chitosan first, gelatin are added, by being chemically crosslinked and being freeze-dried obtained plural gel.Preparation process of the present invention is simple, and raw material is simple, at low cost, and plural gel obtained has preferable bioactivity, can be used for loading cells, while may have anti-microbial property, in terms of this NEW TYPE OF COMPOSITE gel can be preferably applied for repair of cartilage.
Description
Technical field
The present invention relates to a kind of preparation methods of hydroxyethyl chitosan plural gel, belong to biomedical material technology neck
Domain.
Background technology
Chitosan is that the chitin being widely present by nature is obtained by deacetylation, this natural polymer tool
It has the excellent performances such as biocompatibility, blood compatibility, safety, microbic resolvability, is prepared one in bio-medical field
The preferable material of kind.Chitosan gel rubber is needed in preparation process through crosslinking, and crosslinking agent usually selects glutaraldehyde or water-soluble
Property carbodiimides, the introducing of crosslinking agent can reduce the cell compatibility of holder.
Gelatin is also commonly used for preparing bio-medical gel, can be used for loading cells with the compound gel for preparing of chitosan, but
When being that the gel prepared with chitosan loads cell, the activity of cell need to be improved.
Invention content
It is an object of the invention to solve the deficiencies in the prior art, a kind of preparation of hydroxyethyl chitosan plural gel is provided
Method, the plural gel have good loading cells performance, cell can be made to adhere to well, be proliferated, can preferably be applied
In terms of repair of cartilage.
Technical solution
A kind of preparation method of hydroxyethyl chitosan plural gel, includes the following steps：
(1) hydroxyethyl chitosan is prepared：Take 10g chitosans be added in the 50wt% sodium hydroxide solutions of 15~18g into
Row alkalization, obtains chitosan solution, after standing 1~3h under the conditions of -20 DEG C, 15~25ml solvent isopropanols is added, stirring rises
Temperature is stirred for being cooled to room temperature to 70~80 DEG C, and the mixed of 130~180ml isopropanols and 15~19ml ethylene chlorhydrins is then added
Solution is closed, after stirring evenly, is washed using hydrochloric acid solution tune pH7, then using absolute ethyl alcohol, then dialyses, finally freeze
Drying is to get hydroxyethyl chitosan
(2) plural gel is prepared：Hydroxyethyl chitosan is dissolved in the acetum of 1v%, hydroxyethyl chitosan is obtained
Solution, a concentration of 2-5wt% of hydroxyethyl chitosan are added gelatin into hydroxyethyl chitosan solution, hydroxyethyl chitosan with it is bright
Glue mass ratio is 1:3~5:1, it is ultrasonically treated after stirring evenly, is then poured into solution in 48 orifice plates, circulating frozen thaws
Processing 3~9 times, be finally putting into freeze drier be freeze-dried to get.
Further, in step (1), the alkalization carries out in -20 DEG C of refrigerators, and alkalization time is more than for 24 hours.
Further, in step (1), concentration of hydrochloric acid solution 0.1M.
Further, in step (1), selection of dialysing is bag filter of the molecular cut off in 10000Da, dialysis time 2d
More than.
Further, in step (2), the circulating frozen defrosting processing refers to：48 orifice plates are put into -20 DEG C of refrigerators and are freezed
24 hours, 12 hours of defrosting under room temperature are then taken out, then place into freezing in -20 DEG C of refrigerators.
Further, in step (2), hydroxyethyl chitosan is 2 with gelatin mass ratio:1.
Further, in step (2), described to be ultrasonically treated using the Ultrasound Instrument that power is 1000W, ultrasonic time does not surpass
Cross 5min.
Beneficial effects of the present invention：Chitosan is carried out hydroxyethylation by the present invention, and the gelatin of special ratios, system is then added
For at plural gel.Compared with prior art, advantage of the invention is that：1. the plural gel have better mechanical property,
Bioactivity and loading cells performance, can be such that cell preferably adheres to, it is allowed preferably to be proliferated；2. whole preparation process is simple
It is easy to operate, it can largely be prepared；3. the high molecular material cost used is relatively low, pollution-free.
Description of the drawings
Fig. 1 is the scanning electron microscope (SEM) photograph of hydroxyethyl chitosan plural gel made from embodiment 1-3；
Fig. 2 is the cell compatibility testing result of hydroxyethyl chitosan plural gel made from embodiment 1-6；
Fig. 3 is that hydroxyethyl chitosan plural gel prepared by embodiment 3 loads the AO colored graphs after osteoblast.
Specific implementation mode
The present invention will be illustrated by attached drawing and example below, but these specific embodiments do not limit in any way
Protection scope of the present invention.Raw material used in the experimental program can be bought in market.
Embodiment 1
A kind of preparation method of hydroxyethyl chitosan plural gel, includes the following steps：
(1) hydroxyethyl chitosan is prepared：It takes 10g chitosans to be added in the 50wt% sodium hydroxide solutions of 16.7g to carry out
Alkalization, the alkalization carry out in -20 DEG C of refrigerators, and alkalization time is for 24 hours, to obtain chitosan solution, stood under the conditions of -20 DEG C
After 1h, be added 20ml solvent isopropanols, stirring be warming up to 75 DEG C, be stirred for being cooled to room temperature, then be added 133ml isopropanols and
After stirring evenly, 3 are washed with 0.1M hydrochloric acid solution tune pH7, then using absolute ethyl alcohol for the mixed solution of 18.3ml ethylene chlorhydrins
It is secondary, it then selects bag filter of the molecular cut off in 10000Da to dialyse two days, is finally freeze-dried to get hydroxyethyl chitosan；
(2) plural gel is prepared：Hydroxyethyl chitosan is dissolved in the acetum of 1v%, hydroxyethyl chitosan is obtained
Solution, a concentration of 2wt% of hydroxyethyl chitosan, gelatin, hydroxyethyl chitosan and gelatin are added into hydroxyethyl chitosan solution
Mass ratio is 1:2, it is ultrasonically treated that (using the Ultrasound Instrument that power is 1000W, ultrasonic time is after stirring evenly
2min), then solution is poured into 48 orifice plates, 48 orifice plates (it is small to be put into freezing 24 in -20 DEG C of refrigerators by circulating frozen defrosting processing
When, 12 hours of defrosting are then taken out under room temperature, freezing in -20 DEG C of refrigerators is then placed into) 3 times, it is dry to be finally putting into freezing
Dry machine be freeze-dried to get.
The scanning electron microscope (SEM) photograph of plural gel made from embodiment 1 is shown in Fig. 1.
Embodiment 2
A kind of preparation method of hydroxyethyl chitosan plural gel, includes the following steps：
(1) hydroxyethyl chitosan is prepared：It takes 10g chitosans to be added in the 50wt% sodium hydroxide solutions of 16.7g to carry out
Alkalization, the alkalization carry out in -20 DEG C of refrigerators, and alkalization time is for 24 hours, to obtain chitosan solution, stood under the conditions of -20 DEG C
After 1h, be added 20ml solvent isopropanols, stirring be warming up to 75 DEG C, be stirred for being cooled to room temperature, then be added 133ml isopropanols and
After stirring evenly, 3 are washed with 0.1M hydrochloric acid solution tune pH7, then using absolute ethyl alcohol for the mixed solution of 18.3ml ethylene chlorhydrins
It is secondary, it then selects bag filter of the molecular cut off in 10000Da to dialyse two days, is finally freeze-dried to get hydroxyethyl chitosan；
(2) plural gel is prepared：Hydroxyethyl chitosan is dissolved in the acetum of 1v%, hydroxyethyl chitosan is obtained
Solution, a concentration of 2wt% of hydroxyethyl chitosan, gelatin, hydroxyethyl chitosan and gelatin are added into hydroxyethyl chitosan solution
Mass ratio is 1:1, it is ultrasonically treated that (using the Ultrasound Instrument that power is 1000W, ultrasonic time is after stirring evenly
2min), then solution is poured into 48 orifice plates, 48 orifice plates (it is small to be put into freezing 24 in -20 DEG C of refrigerators by circulating frozen defrosting processing
When, 12 hours of defrosting are then taken out under room temperature, freezing in -20 DEG C of refrigerators is then placed into) 3 times, it is dry to be finally putting into freezing
Dry machine be freeze-dried to get.
The scanning electron microscope (SEM) photograph of plural gel made from embodiment 2 is shown in Fig. 1, wherein Figure 1A is the plural gel of embodiment 1,
Figure 1B is the plural gel of embodiment 2.
Embodiment 3
A kind of preparation method of hydroxyethyl chitosan plural gel, includes the following steps：
(1) hydroxyethyl chitosan is prepared：It takes 10g chitosans to be added in the 50wt% sodium hydroxide solutions of 16.7g to carry out
Alkalization, the alkalization carry out in -20 DEG C of refrigerators, and alkalization time is for 24 hours, to obtain chitosan solution, stood under the conditions of -20 DEG C
After 1h, be added 20ml solvent isopropanols, stirring be warming up to 75 DEG C, be stirred for being cooled to room temperature, then be added 133ml isopropanols and
After stirring evenly, 3 are washed with 0.1M hydrochloric acid solution tune pH7, then using absolute ethyl alcohol for the mixed solution of 18.3ml ethylene chlorhydrins
It is secondary, it then selects bag filter of the molecular cut off in 10000Da to dialyse two days, is finally freeze-dried to get hydroxyethyl chitosan；
(2) plural gel is prepared：Hydroxyethyl chitosan is dissolved in the acetum of 1v%, hydroxyethyl chitosan is obtained
Solution, a concentration of 2wt% of hydroxyethyl chitosan, gelatin, hydroxyethyl chitosan and gelatin are added into hydroxyethyl chitosan solution
Mass ratio is 2:1, it is ultrasonically treated that (using the Ultrasound Instrument that power is 1000W, ultrasonic time is after stirring evenly
2min), then solution is poured into 48 orifice plates, 48 orifice plates (it is small to be put into freezing 24 in -20 DEG C of refrigerators by circulating frozen defrosting processing
When, 12 hours of defrosting under room temperature are then taken out, freezing in -20 DEG C of refrigerators is then placed into)
3 times, be finally putting into freeze drier be freeze-dried to get.
The scanning electron microscope (SEM) photograph of plural gel made from embodiment 3 is shown in Fig. 1, wherein Figure 1A is the plural gel of embodiment 1,
Figure 1B is the plural gel of embodiment 2, and Fig. 1 C are the plural gel of embodiment 3.
Embodiment 4
A kind of preparation method of hydroxyethyl chitosan plural gel, includes the following steps：
(1) hydroxyethyl chitosan is prepared：It takes 10g chitosans to be added in the 50wt% sodium hydroxide solutions of 16.7g to carry out
Alkalization, the alkalization carry out in -20 DEG C of refrigerators, and alkalization time is for 24 hours, to obtain chitosan solution, stood under the conditions of -20 DEG C
After 1h, be added 20ml solvent isopropanols, stirring be warming up to 75 DEG C, be stirred for being cooled to room temperature, then be added 133ml isopropanols and
After stirring evenly, 3 are washed with 0.1M hydrochloric acid solution tune pH7, then using absolute ethyl alcohol for the mixed solution of 18.3ml ethylene chlorhydrins
It is secondary, it then selects bag filter of the molecular cut off in 10000Da to dialyse two days, is finally freeze-dried to get hydroxyethyl chitosan；
(2) plural gel is prepared：Hydroxyethyl chitosan is dissolved in the acetum of 1v%, hydroxyethyl chitosan is obtained
Solution, a concentration of 2wt% of hydroxyethyl chitosan, gelatin, hydroxyethyl chitosan and gelatin are added into hydroxyethyl chitosan solution
Mass ratio is 3:1, it is ultrasonically treated that (using the Ultrasound Instrument that power is 1000W, ultrasonic time is after stirring evenly
2min), then solution is poured into 48 orifice plates, 48 orifice plates (it is small to be put into freezing 24 in -20 DEG C of refrigerators by circulating frozen defrosting processing
When, 12 hours of defrosting are then taken out under room temperature, freezing in -20 DEG C of refrigerators is then placed into) 3 times, it is dry to be finally putting into freezing
Dry machine be freeze-dried to get.
Embodiment 5
A kind of preparation method of hydroxyethyl chitosan plural gel, includes the following steps：
(1) hydroxyethyl chitosan is prepared：It takes 10g chitosans to be added in the 50wt% sodium hydroxide solutions of 16.7g to carry out
Alkalization, the alkalization carry out in -20 DEG C of refrigerators, and alkalization time is for 24 hours, to obtain chitosan solution, stood under the conditions of -20 DEG C
After 1h, be added 20ml solvent isopropanols, stirring be warming up to 75 DEG C, be stirred for being cooled to room temperature, then be added 133ml isopropanols and
After stirring evenly, 3 are washed with 0.1M hydrochloric acid solution tune pH7, then using absolute ethyl alcohol for the mixed solution of 18.3ml ethylene chlorhydrins
It is secondary, it then selects bag filter of the molecular cut off in 10000Da to dialyse two days, is finally freeze-dried to get hydroxyethyl chitosan；
(2) plural gel is prepared：Hydroxyethyl chitosan is dissolved in the acetum of 1v%, hydroxyethyl chitosan is obtained
Solution, a concentration of 2wt% of hydroxyethyl chitosan, gelatin, hydroxyethyl chitosan and gelatin are added into hydroxyethyl chitosan solution
Mass ratio is 4:1, it is ultrasonically treated that (using the Ultrasound Instrument that power is 1000W, ultrasonic time is after stirring evenly
2min), then solution is poured into 48 orifice plates, 48 orifice plates (it is small to be put into freezing 24 in -20 DEG C of refrigerators by circulating frozen defrosting processing
When, 12 hours of defrosting under room temperature are then taken out, freezing in -20 DEG C of refrigerators is then placed into)
3 times, be finally putting into freeze drier be freeze-dried to get.
Embodiment 6
A kind of preparation method of hydroxyethyl chitosan plural gel, includes the following steps：
(1) hydroxyethyl chitosan is prepared：It takes 10g chitosans to be added in the 50wt% sodium hydroxide solutions of 16.7g to carry out
Alkalization, the alkalization carry out in -20 DEG C of refrigerators, and alkalization time is for 24 hours, to obtain chitosan solution, stood under the conditions of -20 DEG C
After 1h, be added 20ml solvent isopropanols, stirring be warming up to 75 DEG C, be stirred for being cooled to room temperature, then be added 133ml isopropanols and
After stirring evenly, 3 are washed with 0.1M hydrochloric acid solution tune pH7, then using absolute ethyl alcohol for the mixed solution of 18.3ml ethylene chlorhydrins
It is secondary, it then selects bag filter of the molecular cut off in 10000Da to dialyse two days, is finally freeze-dried to get hydroxyethyl chitosan；
(2) plural gel is prepared：Hydroxyethyl chitosan is dissolved in the acetum of 1v%, hydroxyethyl chitosan is obtained
Solution, a concentration of 2wt% of hydroxyethyl chitosan, gelatin, hydroxyethyl chitosan and gelatin are added into hydroxyethyl chitosan solution
Mass ratio is 5:1, it is ultrasonically treated that (using the Ultrasound Instrument that power is 1000W, ultrasonic time is after stirring evenly
2min), then solution is poured into 48 orifice plates, 48 orifice plates (it is small to be put into freezing 24 in -20 DEG C of refrigerators by circulating frozen defrosting processing
When, 12 hours of defrosting are then taken out under room temperature, freezing in -20 DEG C of refrigerators is then placed into) 3 times, it is dry to be finally putting into freezing
Dry machine be freeze-dried to get.
Performance test
1. hydroxyethyl chitosan plural gel made from couple embodiment 1-6 carries out cell compatibility detection.
Cell compatibility testing process is as follows：Sample is carried out disinfection with 75% alcohol, it is then dry thin with medulla mesenchyma
Born of the same parents cultivate, and are evaluated using CCK-8.Fig. 2 is the cell compatibility test of compound bone holder material prepared by the present invention
As a result.As seen from Figure 2 cell on plural gel holder as time increases, cell survival rate be in increase trend, this
Illustrate that the plural gel prepared has good cell compatibility.
2. the cell proliferation performance detection of plural gel.
Plural gel prepared by embodiment 3 is loaded to the AO colored graphs after osteoblast and sees that Fig. 3, Fig. 3 A are on day 1
AO colored graphs, Fig. 3 B are the 3rd day AO colored graphs, by Fig. 3, it is apparent that cell quantity obviously increases.
Claims (7)
1. a kind of preparation method of hydroxyethyl chitosan plural gel, which is characterized in that include the following steps：
(1) hydroxyethyl chitosan is prepared：It takes 10g chitosans to be added in the 50wt% sodium hydroxide solutions of 15~18g and carries out alkali
Change, obtain chitosan solution, after standing 1~3h under the conditions of -20 DEG C, 15~25ml solvent isopropanols is added, stirring is warming up to
It 70~80 DEG C, is stirred for being cooled to room temperature, 130~180ml isopropanols is then added and the mixing of 15~19ml ethylene chlorhydrins is molten
Liquid after stirring evenly, is washed using hydrochloric acid solution tune pH7, then using absolute ethyl alcohol, then dialyses, be finally freeze-dried,
Up to hydroxyethyl chitosan；
(2) plural gel is prepared：Hydroxyethyl chitosan is dissolved in the acetum of 1v%, it is molten to obtain hydroxyethyl chitosan
Liquid, a concentration of 2-5wt% of hydroxyethyl chitosan, gelatin, hydroxyethyl chitosan and gelatin are added into hydroxyethyl chitosan solution
Mass ratio is 1:3~5:1, it is ultrasonically treated after stirring evenly, is then poured into solution in 48 orifice plates, at circulating frozen defrosting
Reason 3~9 times, be finally putting into freeze drier be freeze-dried to get.
2. the preparation method of hydroxyethyl chitosan plural gel as described in claim 1, which is characterized in that in step (1), institute
It states alkalization to carry out in -20 DEG C of refrigerators, alkalization time is more than for 24 hours.
3. the preparation method of hydroxyethyl chitosan plural gel as described in claim 1, which is characterized in that in step (1), salt
Acid solutions are 0.1M.
4. the preparation method of hydroxyethyl chitosan plural gel as described in claim 1, which is characterized in that in step (1), thoroughly
What analysis was selected is bag filter of the molecular cut off in 10000Da, and dialysis time is 2d or more.
5. the preparation method of hydroxyethyl chitosan plural gel as described in claim 1, which is characterized in that in step (2), institute
Stating circulating frozen defrosting processing refers to：48 orifice plates are put into -20 DEG C of refrigerators and are freezed 24 hours, then takes out and thaws under room temperature
Then 12 hours place into freezing in -20 DEG C of refrigerators.
6. the preparation method of hydroxyethyl chitosan plural gel as described in claim 1, which is characterized in that in step (2), hydroxyl
Ethyl chitosan is 2 with gelatin mass ratio:1.
7. such as the preparation method of claim 1 to 6 any one of them hydroxyethyl chitosan plural gel, which is characterized in that step
Suddenly in (2), described to be ultrasonically treated using the Ultrasound Instrument that power is 1000W, ultrasonic time is no more than 5min.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114344565A (en) * | 2022-01-04 | 2022-04-15 | 陕西科技大学 | Chitosan-gelatin bone repair stent and preparation method thereof |
| CN117357700A (en) * | 2023-12-06 | 2024-01-09 | 上海威高医疗技术发展有限公司 | Hyaluronic acid gel and preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104771779A (en) * | 2015-04-30 | 2015-07-15 | 河南省超亚医药器械有限公司 | Functional dressing and preparation method thereof |
| CN106188442A (en) * | 2016-08-02 | 2016-12-07 | 西安交通大学 | A kind of chitosan derivatives hydrogel and preparation method thereof |
| CN106310350A (en) * | 2016-10-28 | 2017-01-11 | 中国药科大学 | Hydrogel woundplast containing traditional Chinese medicine extracts and preparation method thereof |
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2018
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104771779A (en) * | 2015-04-30 | 2015-07-15 | 河南省超亚医药器械有限公司 | Functional dressing and preparation method thereof |
| CN106188442A (en) * | 2016-08-02 | 2016-12-07 | 西安交通大学 | A kind of chitosan derivatives hydrogel and preparation method thereof |
| CN106310350A (en) * | 2016-10-28 | 2017-01-11 | 中国药科大学 | Hydrogel woundplast containing traditional Chinese medicine extracts and preparation method thereof |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114344565A (en) * | 2022-01-04 | 2022-04-15 | 陕西科技大学 | Chitosan-gelatin bone repair stent and preparation method thereof |
| CN117357700A (en) * | 2023-12-06 | 2024-01-09 | 上海威高医疗技术发展有限公司 | Hyaluronic acid gel and preparation method and application thereof |
| CN117357700B (en) * | 2023-12-06 | 2024-03-22 | 上海威高医疗技术发展有限公司 | Hyaluronic acid gel and preparation method and application thereof |
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