CN109647298A - Polyethylene-zinc oxide micrometer nanometer hierarchical structure composite micro-sphere material and application - Google Patents
Polyethylene-zinc oxide micrometer nanometer hierarchical structure composite micro-sphere material and application Download PDFInfo
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- CN109647298A CN109647298A CN201910097691.4A CN201910097691A CN109647298A CN 109647298 A CN109647298 A CN 109647298A CN 201910097691 A CN201910097691 A CN 201910097691A CN 109647298 A CN109647298 A CN 109647298A
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
- B01J13/02—Making microcapsules or microballoons
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y40/00—Manufacture or treatment of nanostructures
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/20—Small organic molecules
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/30—Synthetic polymers
Abstract
The invention discloses a kind of polyethylene-zinc oxide micrometer nanometer hierarchical structure composite micro-sphere material and applications, polyethylene of the invention-zinc oxide micrometer nanometer hierarchical structure composite micro-sphere material is to immerse polyethylene microballoon in zinc acetate solution, after surface speckles with zinc acetate, take out microballoon, oxygen processing is played by oxygen plasma cleaning machine, makes microsphere surface combination zinc oxide.Surface is placed in zinc nitrate and pregnancy urotropine mixed liquor with the polyethylene microballoon of zinc oxide, is stirred under 80-100 DEG C of water bath with thermostatic control, after complete reaction, washing, it is dry to get.The micro-sphere structure stabilization, size uniformity, diameter are 150-400 μm, and zinc oxide nano rod length is 10-50nm.Human adipose mesenchymal stem cells are seeded on the microballoon, the proliferation of stem cell can be promoted, and maintain the stemness of stem cell.
Description
Technical field
The present invention relates to biomedical engineering technology fields, and in particular to a kind of polyethylene-zinc oxide micrometer nanometer hierarchical
Structure composite micro-sphere material and its application in maintenance stem cell stemness.
Background technique
Stem-cell therapy technology is in gene therapy, autoimmune disease, brain and nervous system disease, angiocarpy
Huge potential applicability in clinical practice is shown in disease treatment.Wherein, the advantage of autologous adult stem cells transplanting is especially prominent.
Adult stem cell refers to the neoblast being present in a kind of differentiated tissue.Under given conditions, adult
Stem cell perhaps generates new stem cell or breaks up by certain program, generates each species specific cell type.It is dry with embryo
Cell is compared, and adult stem cell has many advantages, such as: (1) due to the major histocompatibility complex of each individual
(MHC) different, allogeneic embryonic stem cell and its differentiated tissue's cell are used for clinic and can cause immunological rejection, thus limit
Its clinical application；And adult stem cell can be obtained from patient itself, the problem of histocompatbility may be not present, when treatment, can avoid
Injury of the immunosuppressor to patient is used for a long time.(2) although embryonic stem cell can be divided into various cell types, this point
Change is " non-locating ", not can control embryonic stem cell still at present and is divided into corresponding cell at specific position, current
Way is easy to cause teratoma；And the above problem is not present in application in adult stem cell.However adult stem cell is in body
Limited amount needs amplification in vitro to meet clinical demand.There are contact inhibition phenomenon, cell proliferation rates for traditional plate culture
It is low, and easily differentiation or aging, seriously affect the quality and quantity of stem cell.Therefore, a kind of novel maintenance adult is developed
The cultural method of stem cell stemness is extremely urgent.
Microballoon refers to be made of natural or synthetic high molecular material (such as gelatin, chitosan, polylactic acid), and partial size is in 1-
Between 1000 microns.The preparation method of microballoon mainly has emulsification/solvent evaporation method, spray drying process, supercritical carbon dioxide process
And microfluidic method etc..The difference of preparation method causes the pattern for the microballoon being prepared, properity etc. all to have differences.
Polyethylene (PE) is the polymer as made of vinyl polymerization, has excellent resistance to low temperature, and chemical stability is good,
It is widely used in manufacturing film, hollow product, fiber and daily use Sundry goods etc..Nano zine oxide have excellent skin effect,
Small-size effect and macro quanta tunnel effect etc., while there is photochemical effect and preferably cover imparted energy, have
Wide application prospect.But since nano zine oxide has the characteristics that large specific surface area and specific surface area can be big, itself is easy to reunite；
On the other hand, surface of nanometer zinc oxide polarity is stronger, is not easy in organic media evenly dispersed, this just significantly limits it and receives
The performance of meter Xiao Ying.It yet there are no the form that polyethylene and nano zine oxide are combined to microballoon.
In addition, microballoon is mainly used for the fields such as medicament slow release, cytoskeleton and cell infusion at present, does not have also and utilize microballoon
Promote adult stem cell proliferation and maintains the report of adult stem cell stemness.
Summary of the invention
For the above-mentioned prior art, the object of the present invention is to provide a kind of polyethylene-zinc oxide micrometer nanometer hierarchical structures
Composite microsphere material.Polyethylene and zinc oxide are combined micro-sphere material by novelty of the invention, polyethylene in the micron-scale
Microsphere surface is coated with nano structure of zinc oxide, forms micron/nano multilevel structure composite microsphere material.
Polyethylene of the invention-zinc oxide micrometer nanometer hierarchical structure composite micro-sphere material has good cytocompatibility
Property, adult stem cell proliferation can be promoted, cell survival rate can maintain the stemness of adult stem cell 98% or more, use
Adult stem cell after polyethylene of the invention-zinc oxide micrometer nanometer hierarchical structure composite micro-sphere material amplification has good
Differentiation potential can be divided into skeletonization, fat and cartilage cell.
To achieve the above object, the present invention adopts the following technical scheme:
The first aspect of the present invention provides a kind of polyethylene-zinc oxide micrometer nanometer hierarchical structure composite micro-sphere material
Preparation method, comprising the following steps:
(1) polyethylene microballoon is immersed in zinc acetate solution, reacts 4-24h in 20-30 DEG C of water bath with thermostatic control, takes out polyethylene
Microballoon is handled (vacuum degree 50Pa, processing power 100W, 10 points of processing to polyethylene microballoon using oxygen plasma cleaning machine
Clock), so that polyethylene microsphere surface is covered zinc oxide film；
(2) the polyethylene microballoon of surface covering zinc oxide film is placed in zinc nitrate-hexamethylenetetramine mixed liquor, in
It stirs, reacts 1-4 days under 80-100 DEG C of water bath with thermostatic control, harvest microballoon, washing is dry to get polyethylene-zinc oxide micrometer nanometer
Multilevel structure composite microsphere material.
In step (1), the diameter of the polyethylene microballoon is 150-400 μm.The polyethylene microballoon is commercially available poly- second
Alkene microballoon；Or the polyethylene microballoon that according to prior art prepared by method.As long as the diameter of polyethylene microballoon is at 150-400 μm
Between be used equally for the present invention.
In step (1), the concentration of the zinc acetate solution is 0.01mol/L-0.25mol/L.
In step (2), in the zinc nitrate-hexamethylenetetramine mixed liquor, the quality of zinc nitrate and hexamethylenetetramine
Than for 1:1, the final concentration of 0.01mol/L-0.2mol/L of zinc nitrate.
In step (2), the speed of stirring is 100rpm-1000rpm.
The second aspect of the present invention provides polyethylene-zinc oxide micrometer nanometer hierarchical structure composite of above method preparation
Micro-sphere material.The polyethylene-zinc oxide micrometer nanometer hierarchical structure composite micro-sphere material diameter is 150-400 μm, oxidation
Zinc nanorod length is 10-50nm.
The third aspect of the present invention provides above-mentioned polyethylene-zinc oxide micrometer nanometer hierarchical structure composite micro-sphere material and exists
Following 1) -3) application at least one of:
1) promote adult stem cell proliferation and maintain adult stem cell stemness；
2) amplification in vitro of adult stem cell；
3) preparation promotes adult stem cell proliferation and maintains the culture medium of adult stem cell stemness.
The fourth aspect of the present invention, adult stem cell proliferation can be promoted and maintain adult stem cell stemness by providing one kind
Culture medium, the culture medium with above-mentioned polyethylene-zinc oxide micrometer nanometer hierarchical structure composite micro-sphere material be effectively at
Point.
The fifth aspect of the present invention provides a kind of method of amplification in vitro adult stem cell, comprising the following steps: by adult
Stem cell is seeded in above-mentioned culture medium, carries out amplification cultivation.
Beneficial effects of the present invention:
(1) present invention is for the first time basic unit with polyethylene microballoon, in its surface synthesizing zinc oxide nanostructure, preparation
Composite microsphere material uniform particle diameter, form are controllable.
(2) polyethylene of the invention-zinc oxide micrometer nanometer hierarchical structure composite micro-sphere material has good cell phase
Capacitive can promote adult stem cell proliferation, and can maintain the stemness of adult stem cell, using polyethylene-oxidation of the invention
Adult stem cell after the amplification of zinc micron/nano multilevel structure composite microsphere material has good differentiation potential, can be divided into
Skeletonization, fat and cartilage cell.
Detailed description of the invention:
Fig. 1: polyethylene of the invention-zinc oxide micrometer nanometer hierarchical structure composite micro-sphere material preparation flow figure.
Fig. 2: polyethylene of the invention-zinc oxide micrometer nanometer hierarchical structure composite micro-sphere material structural schematic diagram.
Fig. 3: CCK8 Activity determination shows the proliferation rate of source of people fat mesenchymal stem cell.Wherein, normal group is to be grown in
Cell in culture dish；Material group 1-3 is respectively the polyethylene-zinc oxide micrometer nanometer hierarchical for being grown in embodiment 1-3 acquisition
The cell of structure composite microballoon.
Specific embodiment
It is noted that following detailed description is all illustrative, it is intended to provide further instruction to the application.Unless another
It indicates, all technical and scientific terms used herein has usual with the application person of an ordinary skill in the technical field
The identical meanings of understanding.
As background technology part introduction, existing adult stem cell amplification in vitro method, cell proliferation rate is low, and
Easily differentiation or aging, seriously affects the quality and quantity of adult stem cell during Multiplying culture.
Based on this, novel it can promote adult stem cell proliferation the object of the present invention is to provide a kind of and can maintain to do
The cultural method of cell stemness.
Traditional microballoon is generally used for the fields such as medicament slow release, cytoskeleton and cell infusion, especially as drug
Carrier, the also not report about microballoon in stem cells hyperplasia and stem cell stemness maintain at present.Distinct methods are prepared into
To pattern, the properity of microballoon etc. can all have larger difference, the microballoon of polyethylene and the compound preparation of zinc oxide is current
It yet there are no report.
For the first time the microballoon that pan coating has nano structure of zinc oxide is prepared in polyethylene and zinc oxide by the present invention, and is anticipated
Outer discovery, can using the polyethylene of specific morphology and structure composition-zinc oxide micrometer nanometer hierarchical structure composite micro-sphere material
To promote the proliferation of adult stem cell, and maintain the stemness of stem cell.
In one embodiment of the present invention, polyethylene-zinc oxide micrometer nanometer hierarchical structure composite microballoon is given
The preparation method (Fig. 1) of material, comprising the following steps:
Polyethylene microballoon is immersed in zinc acetate solution, is impregnated 4-24 hours under 20-30 DEG C of water bath with thermostatic control, it is sharp after collection
It is handled with oxygen plasma, microsphere surface is made to adhere to zinc oxide.After to be dried, which is placed in containing zinc nitrate and six methines
Tetramine mass ratio is to stir under 90 DEG C of waters bath with thermostatic control in the solution of 1:1, is reacted 1-4 days, and microballoon is harvested, clear with deionized water
It washes three times, then with after washes of absolute alcohol, vacuum drying obtains the polyethylene microballoon (figure of pan coating nano structure of zinc oxide
2)。
In polyethylene of the invention-zinc oxide micrometer nanometer hierarchical structure composite micro-sphere material preparation method, each step
Between complement each other, have collaboration facilitation, be an organic whole.Wherein, first by the polylactic acid with micron-scale
Microballoon immerses in zinc acetate solution, and is handled using oxygen plasma, and microsphere surface is made to adhere to zinc oxide, and zinc oxide at this time does not have also
It is grown to serve as nanostructure, is intended only as seed layer, on this basis, the present invention is further by surface attachment zinc oxide
Polyethylene microballoon is placed in the mixed solution containing zinc nitrate and hexamethylenetetramine and is handled, with this condition, zinc oxide
Nano bar-shape structure is grown to serve as in polyethylene microsphere surface.Zinc acetate solution concentration and microballoon are in zinc nitrate and six methines
The factors such as reaction temperature and time in tetramine mixed liquor can all influence structure, pattern and the property of the composite microsphere material of preparation
Energy.
Experiment proves that the size of the size of polyethylene microballoon and the nano zine oxide in the growth of polyethylene microsphere surface
It will affect the cultivation effect to adult stem cell, only using the method for the present invention preparation with specific structure composition and pattern
Polyethylene-zinc oxide micrometer nanometer hierarchical structure composite micro-sphere material can just promote the proliferation of adult stem cell, and remain dry
The stemness of cell.
In order to enable those skilled in the art can clearly understand the technical solution of the application, below with reference to tool
The technical solution of the application is described in detail in the embodiment of body.
Test material used in the embodiment of the present invention is the test material of this field routine, can pass through commercial channel
It is commercially available.
Embodiment 1: polyethylene-zinc oxide micrometer nanometer hierarchical structure composite micro-sphere material preparation
It is in 0.15mol/L zinc acetate solution, in 25 DEG C of constant temperature that polyethylene microballoon (150 μm of microsphere diameter), which is immersed concentration,
Water-bath 12h makes polyethylene microsphere surface be coated with zinc acetate；Polyethylene microballoon is taken out, oxygen plasma cleaning machine pair is utilized
Polyethylene microballoon is handled (vacuum degree 50Pa, processing power 100W are handled 10 minutes), and polyethylene microsphere surface is made to cover oxygen
Change zinc layers.
The polyethylene microballoon of surface covering zinc oxide film is placed in zinc nitrate-hexamethylenetetramine mixed liquor, the nitre
In sour zinc-hexamethylenetetramine mixed liquor, the mass ratio of zinc nitrate and hexamethylenetetramine is 1:1, zinc nitrate it is final concentration of
0.01mol/L is stirred under 90 DEG C of waters bath with thermostatic control, is reacted 2 days, is harvested microballoon, is cleaned three times with deionized water, then with anhydrous second
After alcohol cleaning, it is dried in vacuo to get polyethylene-zinc oxide micrometer nanometer hierarchical structure composite micro-sphere material.
Embodiment 2: polyethylene-zinc oxide micrometer nanometer hierarchical structure composite micro-sphere material preparation
It is in 0.01mol/L zinc acetate solution, in 30 DEG C of constant temperature that polyethylene microballoon (400 μm of microsphere diameter), which is immersed concentration,
Water-bath 4h makes polyethylene microsphere surface be coated with zinc acetate；Polyethylene microballoon is taken out, using oxygen plasma cleaning machine to poly-
Ethylene microballoon is handled (vacuum degree 50Pa, processing power 100W are handled 10 minutes), makes the covering oxidation of polyethylene microsphere surface
Zinc layers.
The polyethylene microballoon of surface covering zinc oxide film is placed in zinc nitrate-hexamethylenetetramine mixed liquor, the nitre
In sour zinc-hexamethylenetetramine mixed liquor, the mass ratio of zinc nitrate and hexamethylenetetramine is 1:1, zinc nitrate it is final concentration of
0.1mol/L is stirred under 80 DEG C of waters bath with thermostatic control, is reacted 4 days, is harvested microballoon, is cleaned three times with deionized water, then with anhydrous second
After alcohol cleaning, it is dried in vacuo to get polyethylene-zinc oxide micrometer nanometer hierarchical structure composite micro-sphere material.
Embodiment 3: polyethylene-zinc oxide micrometer nanometer hierarchical structure composite micro-sphere material preparation
It is in 0.25mol/L zinc acetate solution, in 20 DEG C of constant temperature that polyethylene microballoon (200 μm of microsphere diameter), which is immersed concentration,
Water-bath for 24 hours, makes polyethylene microsphere surface be coated with zinc acetate；Polyethylene microballoon is taken out, oxygen plasma cleaning machine pair is utilized
Polyethylene microballoon is handled (vacuum degree 50Pa, processing power 100W are handled 10 minutes), and polyethylene microsphere surface is made to cover oxygen
Change zinc layers.
The polyethylene microballoon of surface covering zinc oxide film is placed in zinc nitrate-hexamethylenetetramine mixed liquor, the nitre
In sour zinc-hexamethylenetetramine mixed liquor, the mass ratio of zinc nitrate and hexamethylenetetramine is 1:1, zinc nitrate it is final concentration of
0.2mol/L is stirred under 100 DEG C of waters bath with thermostatic control, is reacted 1 day, is harvested microballoon, is cleaned three times with deionized water, then with anhydrous second
After alcohol cleaning, it is dried in vacuo to get polyethylene-zinc oxide micrometer nanometer hierarchical structure composite micro-sphere material.
Test example 1:
Source of people fat mesenchymal stem cell is seeded in polyethylene-zinc oxide micrometer prepared by embodiment 1- embodiment 3 to receive
On rice multilevel structure composite microsphere material, after cultivating 1,3,5 day respectively, cell survival rate is detected.Dyed by CCK8, discovery with
Control material (culture dish) is compared, and the cell proliferation rate grown on microballoon significantly increases (Fig. 3), and it is good to illustrate that material has
Biocompatibility, and promote the proliferation of stem cell.Under the action of skeletonization and adipogenic induction liquid, filled between the fat harvested on microballoon
Matter stem cell can be divided into skeletonization and fat cell.
The result shows that polyethylene prepared by the present invention-zinc oxide micrometer nanometer hierarchical structure composite micro-sphere material can promote
Into the proliferation of fat mesenchymal stem cell, and it is able to maintain that the stemness of cell.
The foregoing is merely preferred embodiment of the present application, are not intended to limit this application, for the skill of this field
For art personnel, various changes and changes are possible in this application.Within the spirit and principles of this application, made any to repair
Change, equivalent replacement, improvement etc., should be included within the scope of protection of this application.
Claims (10)
1. a kind of polyethylene-zinc oxide micrometer nanometer hierarchical structure composite micro-sphere material preparation method, which is characterized in that including
Following steps:
(1) polyethylene microballoon is immersed in zinc acetate solution, reacts 4-24h in 20-30 DEG C of water bath with thermostatic control, it is micro- takes out polyethylene
Ball is handled polyethylene microballoon using oxygen plasma cleaning machine, and polyethylene microsphere surface is made to cover zinc oxide film；
(2) the polyethylene microballoon of surface covering zinc oxide film is placed in zinc nitrate-hexamethylenetetramine mixed liquor, in 80-100
It stirs, reacts 1-4 days under DEG C water bath with thermostatic control, harvest microballoon, washing is dried to get polyethylene-zinc oxide micrometer nanometer hierarchical knot
Structure composite microsphere material.
2. preparation method according to claim 1, which is characterized in that in step (1), the diameter of the polyethylene microballoon is
30-400μm。
3. preparation method according to claim 1, which is characterized in that in step (1), the concentration of the zinc acetate solution is
0.01mol/L-0.25mol/L。
4. preparation method according to claim 1, which is characterized in that in step (2) ,-six methine four of zinc nitrate
In amine mixed liquor, the mass ratio of zinc nitrate and hexamethylenetetramine is 1:1, the final concentration of 0.01mol/L- of zinc nitrate
0.2mol/L。
5. preparation method according to claim 1, which is characterized in that in step (2), the speed of stirring is 100rpm-
1000rpm。
6. polyethylene-zinc oxide micrometer nanometer hierarchical structure composite microballoon of the described in any item method preparations of claim 1-5
Material.
7. polyethylene according to claim 6-zinc oxide micrometer nanometer hierarchical structure composite micro-sphere material, feature exist
In the polyethylene-zinc oxide micrometer nanometer hierarchical structure composite micro-sphere material diameter is 150-400 μm, zinc-oxide nano
Stick length is 10-50nm.
8. polyethylene described in claim 6 or 7-zinc oxide micrometer nanometer hierarchical structure composite micro-sphere material is following 1)-3)
Application at least one:
1) promote adult stem cell proliferation and maintain adult stem cell stemness；
2) amplification in vitro of adult stem cell；
3) preparation promotes adult stem cell proliferation and maintains the cultivating system of adult stem cell stemness.
9. one kind can promote adult stem cell proliferation and maintain the cultivating system of adult stem cell stemness, which is characterized in that institute
It is effective that cultivating system, which is stated, with polyethylene described in claim 6 or 7-zinc oxide micrometer nanometer hierarchical structure composite micro-sphere material
Ingredient.
10. a kind of method of amplification in vitro adult stem cell, which comprises the following steps: adult stem cell to be inoculated with
To in cultivating system as claimed in claim 9, amplification cultivation is carried out.
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| CN110684727A (en) * | 2019-10-12 | 2020-01-14 | 山东大学 | Zinc oxide nanorod array cell culture substrate and preparation method and application thereof |
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