CN108434522A - A kind of preparation method of the degradable biocompatibility aquagel membrane of surface layer embedding cell - Google Patents
A kind of preparation method of the degradable biocompatibility aquagel membrane of surface layer embedding cell Download PDFInfo
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Abstract
The present invention provides a kind of preparation methods of the degradable biocompatibility aquagel membrane of surface layer embedding cell.First by natural polymers, pore-foaming agent is soluble in water to obtain casting solution A, and cell is distributed to the casting solution B obtained in casting solution A.With coiling diameter it is d on A glass platesAThe glass bar of copper wire casting solution A is scraped uniformly, with coiling diameter be d on B glass platesBThe glass bar of copper wire casting solution B is scraped uniformly, glass plate B is tipped upside down on glass plate A immediately after, is separated with the copper wire of a diameter of d between two pieces of glass plates, wherein d≤dA+dB.Two pieces of glass plates are soaked into cross-linking agent solution after keeping natural polymers full cross-linked, pore-foaming agent is taken off with massive laundering, obtains a kind of degradable biocompatibility aquagel membrane of surface layer embedding cell.The aquagel membrane thickness is controllable, and good biocompatibility is biodegradable, has a good application prospect in field of tissue engineering technology.
Description
Technical field
The present invention relates to a kind of preparation methods of the degradable biocompatibility aquagel membrane of surface layer embedding cell, belong to raw
Object material and field of tissue engineering technology.
Background technology
Organizational project is to establish the three dimensions complex of cell and biomaterial, that is, has vital living tissue,
To the reconstruction to disease damage tissue progress form, structure and function and reach permanent replacement.The three elements of organizational project include
Seed cell, host material and regulatory factor.Past tissue engineering bracket is usually from the angle design of materialogy, rather than from life
Object angle considers how that so that implantation material is integrated rebuilds tissue, and implantation material is often because cell utilization rate is low, scaffold degradation causes
The factors such as fibrosis, rejection and inflammatory reaction expected repairing effect is not achieved.Natural biologic material derives from a wealth of sources, is nontoxic, is raw
Object compatibility and biodegradability are good, but its processing extraction is difficult, mechanical property is bad, limits it in organizational project
In application.Artificial synthesized inorganic material chemistry property is stablized, good biocompatibility, but degradation rate is slow, Partial digestion production
Object is not easy to be absorbed by organisms, poor mechanical property.Artificial synthesized high-molecular organic material degradation is controllable, but material surface lacks
Cell recognition signal is unfavorable for the activation of the specific adhesion and specific gene of cell.
The multicomponent system that macromolecule hydrogel is made of polymer three-dimensional network and water, produces environmental stimulus in shape
The polymer network of raw great variety.Hydrogel is a kind of high molecular material similar to vital tissues, hydrogel with many groups
It knits very close with extracellular matrix, shows good biocompatibility, rejection will not be caused in vivo by being put into, and be simultaneously
Nutriment transports and the discharge of metabolic waste provides better channels.Hydrogel can be used as permanent soft tissue injury substitute, or
As cell culture medium material.Chaudhuri etc. is by the hydrogel containing cartilage cell for substituting damaged cartilage【Nature
Materials, 2017,16：1243-1251】.Professor Chang Jiang etc. is prepared for the CaAlg/CaSiO of injectable3@SiO2Compound water congealing
Glue, the hydrogel can promote rat bone marrow mesenchymal stem cells Osteoblast Differentiation【Acta Biomaterialia, 2013,9
(11)：9107-9117】.
Degradable hydrogel can be under organism physiology environment, and by hydrolyzing, digesting the effects that is degraded into harmless to body
Small-molecule substance, and these small molecule catabolites are typically that itself there is in vivo, such as amino acid, lactic acid, most
Afterwards, it fully absorbs and drains by the metabolism of body, have no toxic side effect to body, thus have extensively in biomedical sector
Application.It includes agar, sodium alginate, gelatin, chitosan, fiber to study more natural polymer degradable hydrogel at present
Element and carboxymethyl chitosan etc..But since the smooth surface of hydrogel and high hydrophily, cell are not easy in hydrogel surface
Adherency proliferation.Even if adding fibronectin splicing variants in hydrogel promotes cell adherence, it is also difficult to control the close of attached cell
Degree.Dimensional culture causes cell death because the too deep influence nutrient of cell embedding and metabolin transmit.
Invention content
In view of the deficiencies of the prior art, the technical issues of present invention intends to solve is that cell is not easy to adhere in hydrogel surface,
It is difficult to control attached cell density, dimensional culture causes cell death because the too deep influence nutrient of cell embedding and metabolin transmit
The problem of.
The present invention solves the cell and is not easy to adhere in hydrogel surface, it is difficult to control attached cell density, dimensional culture
Because the too deep influence nutrient of cell embedding and metabolin transmit, the technical solution for the problem of causing cell death is to provide a kind of surface layer
The preparation method of the degradable biocompatibility aquagel membrane of embedding cell.
The present invention provides a kind of preparation methods of the degradable biocompatibility aquagel membrane of surface layer embedding cell.First
By natural polymers, pore-foaming agent is soluble in water to obtain casting solution A, and cell is distributed to the casting film obtained in casting solution A
Liquid B.With coiling diameter it is d on A glass platesAThe glass bar of copper wire casting solution A is scraped uniformly, with winding on B glass plates
The glass bar of the copper wire of a diameter of dB scrapes casting solution B uniformly, immediately after tips upside down on glass plate B on glass plate A, two pieces of glass
It is separated with the copper wire of a diameter of d between glass plate, wherein d≤dA+dB.Two pieces of glass plates, which are soaked into cross-linking agent solution, to be made naturally
After high molecular polymer is full cross-linked, pore-foaming agent is taken off with massive laundering, obtains a kind of degradable biological of surface layer embedding cell
Biocompatible hydrogel film.Preparation method of the present invention is simple, does not introduce any toxic reagent, and the aquagel membrane thickness is controllable, raw
Object compatibility is good, biodegradable, has a good application prospect in field of tissue engineering technology.
The present invention provides a kind of preparation methods of the degradable biocompatibility aquagel membrane of surface layer embedding cell, special
Sign is to include the following steps：
A) aseptically, first by 0.1-10g natural polymers, 0.05-5g pore-foaming agents are dissolved in 50-
In 100ml deionized waters, stirring and dissolving is uniform, obtains casting solution A；Cell is distributed to the casting solution B obtained in casting solution A,
Casting solution A is positioned over spare in sterile chamber, casting solution B containing cell is now with the current；
B) cross-linking agent aqueous solution that mass percent is 0.5%-20% is prepared, sterilization is placed in standby in sterile chamber
With；
C) aseptically, step a) the casting solution A prepared are poured on the glass plate A of dry cleansing, is with thickness
10-1000 μm of knifing stick A spreads to liquid film A in homogeneous thickness, and step a) the casting solution B prepared are equally poured into dry cleansing
On glass plate B, the knifing stick B for being 10-1000 μm with thickness spreads to liquid film B in homogeneous thickness, is immediately after buckled to glass plate B
It on glass plate A, is separated with 10-2000 μm of copper wire between glass plate A and glass plate B, it is desirable that brass wire diameter is less than or equal to scrape
The sum of the thickness of film stick A and knifing stick B；Since natural polymers have certain viscosity, liquid film A and liquid film B can be temporary
When keep certain thickness, and thickness is determined by knifing stick, and liquid film A melts with liquid film B phases after glass plate B is tipped upside down on glass plate A
Close no interface；
D) aseptically, the liquid film that step c) is obtained is soaked into crosslinking agent together with glass plate A and glass plate B
Make natural polymers full cross-linked in aqueous solution, after taking off pore-foaming agent with massive laundering, obtains a kind of surface layer embedding cell
Degradable biocompatibility aquagel membrane, natural polymers have degradability and good biocompatibility；
E) the aquagel membrane thickness is controllable, and celliferous hydrogel layer thickness is also controllable, and cell is in the surface layer of hydrogel,
It is easy to carry out mass exchange with the external world, avoids the too deep influence nutrient of cell embedding and metabolin transmission, cause cell death
Problem；The hydrogel good biocompatibility of surface layer embedding cell is obtained, hydrogel is biodegradable, has in field of tissue engineering technology
Good application prospect.
Natural polymers of the present invention are arbitrary in agar, sodium alginate, gelatin, carboxymethyl chitosan
One or more kinds of mixtures；Pore-foaming agent of the present invention is in polyvinyl alcohol, polyethylene glycol, povidone, lactose, sucrose
Any one or two or more mixtures；Cell of the present invention is mesenchymal stem cell, cord blood stem cell, soft
Any one or the two or more mixtures of osteocyte, fibroblast, epidermal cell, cardiac muscle cell.As described in claim 1
A kind of preparation method of the degradable biocompatibility aquagel membrane of surface layer embedding cell, it is characterized in that the crosslinking agent is hydrogen
Any one in calcium oxide, calcium chloride, Geniposide, glyoxal, glutaraldehyde or two or more mixtures.
Specific implementation mode
Specific embodiments of the present invention are described below, but the present invention should not be limited by the examples.
Embodiment 1.
A) aseptically, first by 10g agar, 5g polyvinyl alcohol is dissolved in 100ml deionized waters, and stirring and dissolving is equal
It is even, obtain casting solution A；Mesenchymal stem cell is distributed to the casting solution B obtained in casting solution A, casting solution A is positioned over
Spare in sterile chamber, the casting solution B containing mesenchymal stem cell is now with the current；
B) glyoxal water solution that mass percent is 20% is prepared, sterilization is placed in spare in sterile chamber；
C) aseptically, step a) the casting solution A prepared are poured on the glass plate A of dry cleansing, is with thickness
1000 μm of knifing stick A spreads to liquid film A in homogeneous thickness, and step a) the casting solution B prepared are equally poured into the glass of dry cleansing
On glass plate B, the knifing stick B for being 1000 μm with thickness spreads to liquid film B in homogeneous thickness, and glass plate B is tipped upside down on glass immediately after
On glass plate A, separated with 2000 μm of copper wire between glass plate A and glass plate B, it is desirable that brass wire diameter be less than or equal to knifing stick A and
The sum of the thickness of knifing stick B；Since there is agar certain viscosity, liquid film A and liquid film B can temporarily keep certain thickness, and it is thick
Degree is determined that liquid film A and liquid film B blends no interface after tipping upside down on glass plate B on glass plate A by knifing stick；
D) aseptically, the liquid film that step c) is obtained is soaked into glyoxal together with glass plate A and glass plate B
Make agar full cross-linked in aqueous solution, after taking off polyvinyl alcohol with massive laundering, it is dry thin to obtain a kind of surface layer embedding medulla mesenchyma
The degradable biocompatibility aquagel membrane of born of the same parents, agar have degradability and good biocompatibility；
E) the aquagel membrane thickness is controllable, and the hydrogel layer thickness containing mesenchymal stem cell is also controllable, is filled between marrow
Matter stem cell is in the surface layer of hydrogel, is easy to carry out mass exchange with the external world, avoids mesenchymal stem cell and embedded
It is deep to influence nutrient and metabolin transmission, cause the problem of mesenchymal stem cell death；Obtain surface layer embedding medulla mesenchyma
The hydrogel good biocompatibility of stem cell, hydrogel is biodegradable, has a good application prospect in field of tissue engineering technology.
Embodiment 2.
A) aseptically, first by 0.1g sodium alginates, 0.05g polyethylene glycol is dissolved in 50ml deionized waters, stirs
It mixes and is uniformly dissolved, obtain casting solution A；Cord blood stem cell is distributed to the casting solution B obtained in casting solution A, casting solution A is put
It is placed in spare in sterile chamber, the casting solution B containing cord blood stem cell is now with the current；
B) calcium chloride water that mass percent is 0.5% is prepared, sterilization is placed in spare in sterile chamber；
C) aseptically, step a) the casting solution A prepared are poured on the glass plate A of dry cleansing, is with thickness
10 μm of knifing stick A spreads to liquid film A in homogeneous thickness, and step a) the casting solution B prepared are equally poured into the glass of dry cleansing
On plate B, the knifing stick B for being 10 μm with thickness spreads to liquid film B in homogeneous thickness, and glass plate B is tipped upside down on glass plate A immediately after
On, it is separated with 20 μm of copper wire between glass plate A and glass plate B, it is desirable that brass wire diameter is less than or equal to knifing stick A and knifing stick B
The sum of thickness；Since sodium alginate has a certain viscosity, liquid film A and liquid film B can temporarily keep certain thickness, and thickness by
Knifing stick determines that liquid film A and liquid film B blends no interface after tipping upside down on glass plate B on glass plate A；
D) aseptically, the liquid film that step c) is obtained is soaked into calcium chloride together with glass plate A and glass plate B
Make sodium alginate full cross-linked in aqueous solution, after taking off polyethylene glycol with massive laundering, it is dry thin to obtain a kind of surface layer embedding Cord blood
The degradable biocompatibility aquagel membrane of born of the same parents, sodium alginate have degradability and good biocompatibility；
E) the aquagel membrane thickness is controllable, and the hydrogel layer thickness containing cord blood stem cell is also controllable, cord blood stem cell
Surface layer in hydrogel is easy to carry out mass exchange with the external world, avoid cord blood stem cell embed too deep influences nutrient with
Metabolin transmits, and causes the problem of cord blood stem cell death；Obtain the hydrogel biofacies of surface layer embedding cord blood stem cell
Capacitive is good, and hydrogel is biodegradable, has a good application prospect in field of tissue engineering technology.
Embodiment 3.
A) aseptically, first by 5g gelatin, 1g povidone is dissolved in 80ml deionized waters, and stirring and dissolving is uniform,
Obtain casting solution A；Cartilage cell is distributed to the casting solution B obtained in casting solution A, casting solution A is positioned in sterile chamber
Spare, the casting solution B containing cartilage cell is now with the current；
B) the Geniposide aqueous solution that mass percent is 0.8% is prepared, sterilization is placed in spare in sterile chamber；
C) aseptically, step a) the casting solution A prepared are poured on the glass plate A of dry cleansing, is with thickness
500 μm of knifing stick A spreads to liquid film A in homogeneous thickness, and step a) the casting solution B prepared are equally poured into the glass of dry cleansing
On plate B, the knifing stick B for being 500 μm with thickness spreads to liquid film B in homogeneous thickness, and glass plate B is tipped upside down on glass plate immediately after
It on A, is separated with 900 μm of copper wire between glass plate A and glass plate B, it is desirable that brass wire diameter is less than or equal to knifing stick A and knifing stick
The sum of thickness of B；Since gelatin has certain viscosity, liquid film A and liquid film B can temporarily keep certain thickness, and thickness is by scraping
Film stick determines that liquid film A and liquid film B blends no interface after tipping upside down on glass plate B on glass plate A；
D) aseptically, the liquid film that step c) is obtained is soaked into Geniposide together with glass plate A and glass plate B
Make gelatin full cross-linked in aqueous solution, after taking off povidone with massive laundering, obtains a kind of the degradable of surface layer embedding cartilage cell
Biocompatible hydrogel film, gelatin have degradability and good biocompatibility；
E) the aquagel membrane thickness is controllable, and the hydrogel layer thickness containing cartilage cell is also controllable, and cartilage cell is in water-setting
The surface layer of glue is easy to carry out mass exchange with the external world, avoids cartilage cell and embeds too deep influence nutrient and metabolin transmission, draws
Play the problem of cartilage cell's death；The hydrogel good biocompatibility of surface layer embedding cartilage cell is obtained, hydrogel biological can drop
Solution, has a good application prospect in field of tissue engineering technology.
Embodiment 4.
A) aseptically, first by 2g carboxymethyl chitosans, 1g lactose is dissolved in 60ml deionized waters, stirring and dissolving
Uniformly, casting solution A is obtained；Fibroblast is distributed to the casting solution B obtained in casting solution A, casting solution A is positioned over sterile appearance
Spare in device, the casting solution B containing fibroblast is now with the current；
B) glutaraldehyde water solution that mass percent is 10% is prepared, sterilization is placed in spare in sterile chamber；
C) aseptically, step a) the casting solution A prepared are poured on the glass plate A of dry cleansing, is with thickness
200 μm of knifing stick A spreads to liquid film A in homogeneous thickness, and step a) the casting solution B prepared are equally poured into the glass of dry cleansing
On plate B, the knifing stick B for being 200 μm with thickness spreads to liquid film B in homogeneous thickness, and glass plate B is tipped upside down on glass plate immediately after
It on A, is separated with 350 μm of copper wire between glass plate A and glass plate B, it is desirable that brass wire diameter is less than or equal to knifing stick A and knifing stick
The sum of thickness of B；Since there is carboxymethyl chitosan certain viscosity, liquid film A and liquid film B can temporarily keep certain thickness, and
Thickness is determined that liquid film A and liquid film B blends no interface after tipping upside down on glass plate B on glass plate A by knifing stick；
D) aseptically, the liquid film that step c) is obtained is soaked into glutaraldehyde together with glass plate A and glass plate B
Make carboxymethyl chitosan full cross-linked in aqueous solution, after massive laundering demulsification sugar, obtains a kind of surface layer embedding fibroblast
Degradable biocompatibility aquagel membrane, carboxymethyl chitosan have degradability and good biocompatibility；
E) the aquagel membrane thickness is controllable, and the hydrogel layer thickness containing fibroblast is also controllable, and fibroblast is in water-setting
The surface layer of glue is easy to carry out mass exchange with the external world, avoids fibroblast and embeds too deep influence nutrient and metabolin transmission, draws
Play the problem of fibroblast death；The hydrogel good biocompatibility of surface layer embedding fibroblast is obtained, hydrogel biological can drop
Solution, has a good application prospect in field of tissue engineering technology.
Claims (5)
1. a kind of preparation method of the degradable biocompatibility aquagel membrane of surface layer embedding cell, it is characterized in that including following step
Suddenly：
A) aseptically, first by 0.1-10g natural polymers, 0.05-5g pore-foaming agents are dissolved in 50-100ml and go
In ionized water, stirring and dissolving is uniform, obtains casting solution A；Cell is distributed to the casting solution B obtained in casting solution A, by casting solution
A is positioned over spare in sterile chamber, and casting solution B containing cell is now with the current；
B) cross-linking agent aqueous solution that mass percent is 0.5%-20% is prepared, sterilization is placed in spare in sterile chamber；
C) aseptically, step a) the casting solution A prepared are poured on the glass plate A of dry cleansing, is 10- with thickness
1000 μm of knifing stick A spreads to liquid film A in homogeneous thickness, and step a) the casting solution B prepared are equally poured into the glass of dry cleansing
On glass plate B, the knifing stick B for being 10-1000 μm with thickness spreads to liquid film B in homogeneous thickness, immediately after tips upside down on glass plate B
It on glass plate A, is separated with 10-2000 μm of copper wire between glass plate A and glass plate B, it is desirable that brass wire diameter is less than or equal to knifing
The sum of the thickness of stick A and knifing stick B；Since natural polymers have certain viscosity, liquid film A and liquid film B can be temporary
Certain thickness is kept, and thickness is determined by knifing stick, liquid film A and liquid film B is blended after glass plate B is tipped upside down on glass plate A
There is no interface；
D) aseptically, that liquid film that step c) is obtained is soaked into crosslinking agent together with glass plate A and glass plate B is water-soluble
Make natural polymers full cross-linked in liquid, after taking off pore-foaming agent with massive laundering, obtain a kind of surface layer embedding cell can
Degradation biological biocompatible hydrogel film, natural polymers have degradability and good biocompatibility；
E) the aquagel membrane thickness is controllable, and celliferous hydrogel layer thickness is also controllable, and cell is in the surface layer of hydrogel, is easy to
The problem of mass exchange being carried out with the external world, avoiding the too deep influence nutrient of cell embedding and metabolin transmission, cause cell death；
The hydrogel good biocompatibility of surface layer embedding cell is obtained, hydrogel is biodegradable, has in field of tissue engineering technology good
Application prospect.
2. a kind of preparation method of the degradable biocompatibility aquagel membrane of surface layer embedding cell as described in claim 1,
It is characterized in that the natural polymers be agar, sodium alginate, gelatin, in carboxymethyl chitosan any one or
Two or more mixtures.
3. a kind of preparation method of the degradable biocompatibility aquagel membrane of surface layer embedding cell as described in claim 1,
It is characterized in that the pore-foaming agent be polyvinyl alcohol, polyethylene glycol, povidone, lactose, any one or two kinds in sucrose with
Upper mixture.
4. a kind of preparation method of the degradable biocompatibility aquagel membrane of surface layer embedding cell as described in claim 1,
It is characterized in that the cell be mesenchymal stem cell, cord blood stem cell, cartilage cell, fibroblast, epidermal cell,
Any one or the two or more mixtures of cardiac muscle cell.
5. a kind of preparation method of the degradable biocompatibility aquagel membrane of surface layer embedding cell as described in claim 1,
It is characterized in that the crosslinking agent is calcium hydroxide, calcium chloride, Geniposide, glyoxal, any one or two kinds in glutaraldehyde
The above mixture.
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| CN110302425A (en) * | 2019-06-20 | 2019-10-08 | 温州医科大学附属第一医院 | Mix the preparation method and applications of hydrogel biological material |
| CN112263917A (en) * | 2020-10-19 | 2021-01-26 | 天津工业大学 | Oxalic acid and genipin cross-linked hydrogel filtering membrane and application thereof in molecular ion separation under high salinity |
| CN114577983A (en) * | 2022-01-26 | 2022-06-03 | 西安卓恰新材料科技有限公司 | Experimental method and experimental device for in-vitro degradation experiment |
| CN116041779A (en) * | 2023-01-15 | 2023-05-02 | 陕西科技大学 | PH response type macroporous hydrogel loaded with nucleic acid lysate, and preparation method and application thereof |
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| CN116041779B (en) * | 2023-01-15 | 2024-05-07 | 陕西科技大学 | PH response type macroporous hydrogel loaded with nucleic acid lysate, and preparation method and application thereof |
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