CN109851834A - Polyethylene-hydroxyapatite micrometre nanometer hierarchical structure composite micro-sphere material and application - Google Patents
Polyethylene-hydroxyapatite micrometre nanometer hierarchical structure composite micro-sphere material and application Download PDFInfo
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- CN109851834A CN109851834A CN201910097388.4A CN201910097388A CN109851834A CN 109851834 A CN109851834 A CN 109851834A CN 201910097388 A CN201910097388 A CN 201910097388A CN 109851834 A CN109851834 A CN 109851834A
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Abstract
The invention discloses a kind of polyethylene-hydroxyapatite micrometre nanometer hierarchical structure composite micro-sphere material and applications, polyethylene of the invention-hydroxyapatite micrometre nanometer hierarchical structure composite micro-sphere material is to immerse polyethylene microballoon in calcium nitrate-ammonium dihydrogen phosphate mixed liquor, adjust pH to 8-10, it is stirred to react under 40-70 DEG C of water bath with thermostatic control 1-5 days, collect microballoon, washing, it is dry, obtain the polyethylene complex microsphere of pan coating hydroxyapatite nano-structure.The micro-sphere structure stabilization, size uniformity, diameter are 50-400 μm, and hydroxyapatite nanoparticle length is 20-100nm.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-hydroxyapatite micrometre nanometer
Multilevel structure composite microsphere 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..Hydroxyapatite is because having good bio-compatible
Property is widely used today as organizational project, especially orthopaedics correlative study.The acquisition methods of hydroxyapatite mainly have hydro-thermal method,
Chemical deposition, sol-gel method etc., wherein hydro-thermal method needs high-temperature and high-pressure conditions；Sol-gel method needs alkaline condition
And want high-temperature process；These methods are not suitable for growing hydroxyapatite on polyethylene 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-hydroxyapatite micrometre nanometer hierarchicals
Structure composite micro-sphere material.Polyethylene of the invention-hydroxyapatite micrometre nanometer hierarchical structure composite micro-sphere material has good
Good cell compatibility, can promote adult stem cell proliferation, and can maintain the stemness of adult stem cell, be gathered using of the invention
Adult stem cell after ethylene-hydroxyapatite micrometre nanometer hierarchical structure composite micro-sphere material amplification has good differentiation latent
Can, skeletonization, fat and cartilage cell can be divided into.
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-hydroxyapatite micrometre nanometer hierarchical structure composite microballoon material
The preparation method of material, comprising the following steps:
Polyethylene microballoon is immersed in calcium nitrate-ammonium dihydrogen phosphate mixed liquor, pH to 8-10 is adjusted, in 40-70 DEG C of constant temperature
It is stirred to react under water-bath 1-5 days, harvests microballoon, washed, it is dry, that is, it is more that polyethylene-hydroxyapatite micrometre nanometer is prepared
Level structure composite microsphere material.
The diameter of the polyethylene microballoon is 50-400 μm.The polyethylene microballoon is commercially available polyethylene microballoon；Or
The polyethylene microballoon of method preparation according to prior art.As long as the diameter of polyethylene microballoon is used equally between 50-400 μm
The present invention.
In the calcium nitrate-ammonium dihydrogen phosphate mixed liquor, the concentration ratio of calcium nitrate and ammonium dihydrogen phosphate is 3:3-5:3.
PH to 8-10 is adjusted by the way that ammonium hydroxide is added dropwise.Hydroxyapatite has good biocompatibility and bioactivity, with calcium nitrate and
Ammonium dihydrogen phosphate, which carries out reaction, can be prepared hydroxyapatite, but need high-temperature calcination to handle in preparation process.The present invention
It is found surprisingly that, is that 3:3-5:3 is configured to calcium nitrate-diammonium hydrogen phosphate mixed liquor with concentration ratio, by the polyethylene of certain diameter
It is stirred to react under 40-70 DEG C of water bath with thermostatic control in microballoon submergence calcium nitrate-diammonium hydrogen phosphate mixed liquor, the speed of stirring is
100rpm-1000rpm is handled without subsequent high-temperature calcination, so that it may which polyethylene microsphere surface in the micron-scale forms nanometer
The hydroxyapatite of size.
The second aspect of the present invention provides polyethylene-hydroxyapatite micrometre nanometer hierarchical structure of above method preparation
Composite microsphere material.The polyethylene-hydroxyapatite micrometre nanometer hierarchical structure composite micro-sphere material diameter is 50-400 μ
M, hydroxyapatite nanoparticle length are 20-100nm.
The third aspect of the present invention provides above-mentioned polyethylene-hydroxyapatite micrometre nanometer hierarchical structure composite microballoon material
Expect following 1) -3) at least one of in application:
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.
The fourth aspect of the present invention, adult stem cell proliferation can be promoted and maintain adult stem cell stemness by providing one kind
Cultivating system, the culture medium is with above-mentioned polyethylene-hydroxyapatite micrometre nanometer hierarchical structure composite micro-sphere material
Effective component.
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 cultivating system, carries out amplification cultivation.
Beneficial effects of the present invention:
(1) polyethylene of the invention-hydroxyapatite micrometre nanometer hierarchical structure composite micro-sphere material, preparation condition temperature
Be not necessarily to high temperature, reaction under high pressure condition, composite microsphere material uniform particle diameter, the form of preparation be controllable.
(2) polyethylene of the invention-hydroxyapatite micrometre nanometer hierarchical structure composite micro-sphere material has good thin
Cell phase capacitive can promote adult stem cell proliferation, and can maintain the stemness of adult stem cell, using polyethylene-of the invention
Adult stem cell after the amplification of hydroxyapatite micrometre nanometer hierarchical structure composite micro-sphere material has good differentiation potential, can
To be divided into skeletonization, fat and cartilage cell.
Detailed description of the invention:
Fig. 1: polyethylene of the invention-hydroxyapatite micrometre nanometer hierarchical structure composite micro-sphere material preparation flow
Figure.
Fig. 2: polyethylene of the invention-hydroxyapatite micrometre nanometer hierarchical structure composite micro-sphere material structural representation
Figure.
Fig. 3: CCK8 Activity determination result；Wherein, normal group is the cell being grown in culture dish；Material group 1-3 difference
For the polyethylene-hydroxyapatite micrometre nanometer hierarchical structure composite microballoon cell for being grown in embodiment 1-3 acquisition.
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.And the present invention mentions for the first time
Polyethylene-hydroxyapatite micrometre nanometer hierarchical structure composite the micro-sphere material that can use specific morphology and structure composition out comes
Promote the proliferation of adult stem cell, and maintains the stemness of stem cell.
In one embodiment of the present invention, polyethylene-hydroxyapatite micrometre nanometer hierarchical structure composite is given
The preparation method of micro-sphere material (preparation flow is as shown in Figure 1), comprising the following steps:
It is basic unit with polyethylene microballoon, in its surface synthesizing hydroxylapatite nanostructure；Specifically:
Polyethylene microballoon is impregnated in calcium nitrate and ammonium dihydrogen phosphate, in stirring and 40-70 DEG C of water bath with thermostatic control item
Under part, solution ph alkalinity is adjusted by the way that ammonium hydroxide is added dropwise, after reacting 1-5 days, microballoon is collected, is cleaned three times with deionized water,
Again with after washes of absolute alcohol, vacuum drying obtains polyethylene microballoon (such as Fig. 2 of pan coating hydroxyapatite nano-structure
It is shown).
Hydroxyapatite is related because having good biocompatibility to be widely used today as organizational project, especially orthopaedics
Research.The acquisition methods of hydroxyapatite mainly have hydro-thermal method, chemical deposition, sol-gel method etc., wherein hydro-thermal method needs
Want high-temperature and high-pressure conditions；Sol-gel method needs alkaline condition and wants high-temperature process；These methods are not suitable in poly- second
Hydroxyapatite is grown on alkene microballoon.
Hydroxyapatite nano-structure is grown in polyethylene microsphere surface to realize, the present invention is to immerse polyethylene microballoon
It in calcium nitrate-ammonium dihydrogen phosphate mixed liquor, is reacted in water bath with thermostatic control system under alkaline condition, thus in polyethylene microballoon table
Face deposits hydroxyapatite nanoparticle.
The reaction condition of above-mentioned whole preparation process is mild, and polyethylene microballoon does not pass through high temperature melting processing, is still real
The microballoon of the heart.In addition, by control calcium nitrate-ammonium dihydrogen phosphate mixed liquor in the concentration of calcium nitrate and ammonium dihydrogen phosphate ratio and
Reaction condition keeps the composite microsphere material uniform particle diameter of preparation, form controllable.
Experiment proves that only using polyethylene-hydroxyl with specific structure composition and pattern of the method for the present invention preparation
Base apatite micron/nano multilevel structure composite microsphere material can promote the proliferation of adult stem cell, and maintain the dry of stem cell
Property.
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-hydroxyapatite micrometre nanometer hierarchical structure composite micro-sphere material preparation
Polyethylene microballoon (50 μm of microsphere diameter) is immersed into (calcium nitrate and phosphoric acid in calcium nitrate-ammonium dihydrogen phosphate mixed liquor
The concentration ratio of ammonium dihydrogen is 4:3), pH to 9 is adjusted by being added dropwise, is stirred to react under 55 DEG C of waters bath with thermostatic control 3 days, is harvested micro-
Ball is cleaned three times with deionized water, then with after washes of absolute alcohol, polyethylene-hydroxyapatite is prepared in vacuum drying
Micron/nano multilevel structure composite microsphere material.
Embodiment 2: polyethylene-hydroxyapatite micrometre nanometer hierarchical structure composite micro-sphere material preparation
Polyethylene microballoon (400 μm of microsphere diameter) is immersed into (calcium nitrate and phosphoric acid in calcium nitrate-ammonium dihydrogen phosphate mixed liquor
The concentration ratio of ammonium dihydrogen is 5:3), pH to 10 is adjusted by being added dropwise, is stirred to react under 40 DEG C of waters bath with thermostatic control 5 days, is harvested micro-
Ball is cleaned three times with deionized water, then with after washes of absolute alcohol, polyethylene-hydroxyapatite is prepared in vacuum drying
Micron/nano multilevel structure composite microsphere material.
Embodiment 3: polyethylene-hydroxyapatite micrometre nanometer hierarchical structure composite micro-sphere material preparation
Polyethylene microballoon (200 μm of microsphere diameter) is immersed into (calcium nitrate and phosphoric acid in calcium nitrate-ammonium dihydrogen phosphate mixed liquor
The concentration ratio of ammonium dihydrogen is 3:3), pH to 8 is adjusted by being added dropwise, is stirred to react under 70 DEG C of waters bath with thermostatic control 1 day, is harvested micro-
Ball is cleaned three times with deionized water, then with after washes of absolute alcohol, polyethylene-hydroxyapatite is prepared in vacuum drying
Micron/nano multilevel structure composite microsphere material.
Test example 1:
It is micro- that source of people fat mesenchymal stem cell is seeded in polyethylene-hydroxyapatite prepared by embodiment 1- embodiment 3
On rice nanometer hierarchical structure composite micro-sphere material, after cultivating 1,3,5 day respectively, cell survival rate is detected.It is dyed, is sent out by CCK8
Now compared with control material (culture dish), the cell survival rate grown on microballoon is significantly increased, and with incubation time
Extend, cell quantity gradually rises (Fig. 3), illustrates that material has good biocompatibility, and mesenchyma can be promoted dry thin
The proliferation of born of the same parents.Under the action of skeletonization and adipogenic induction liquid, the fat mesenchymal stem cell harvested on microballoon can be divided into
Bone and fat cell.
The result shows that polyethylene prepared by the present invention-hydroxyapatite micrometre nanometer hierarchical structure composite micro-sphere material energy
Enough promote the proliferation of fat mesenchymal stem cell, and 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-hydroxyapatite micrometre nanometer hierarchical structure composite micro-sphere material preparation method, which is characterized in that
The following steps are included:
Polyethylene microballoon is immersed in calcium nitrate-ammonium dihydrogen phosphate mixed liquor, pH to 8-10 is adjusted, in 40-70 DEG C of water bath with thermostatic control
Under be stirred to react 1-5 days, harvest microballoon, wash, it is dry, that is, polyethylene-hydroxyapatite micrometre nanometer hierarchical knot is prepared
Structure composite microsphere material.
2. preparation method according to claim 1, which is characterized in that the diameter of the polyethylene microballoon is 50-400 μm.
3. preparation method according to claim 1, which is characterized in that in the calcium nitrate-ammonium dihydrogen phosphate mixed liquor, nitre
The concentration ratio of sour calcium and ammonium dihydrogen phosphate is 3:3-5:3.
4. preparation method according to claim 1, which is characterized in that adjust pH to 8-10 by the way that ammonium hydroxide is added dropwise.
5. preparation method according to claim 1, which is characterized in that the speed of stirring is 100rpm-1000rpm.
6. polyethylene-hydroxyapatite micrometre nanometer hierarchical structure composite of the described in any item method preparations of claim 1-5
Micro-sphere material.
7. polyethylene according to claim 6-hydroxyapatite micrometre nanometer hierarchical structure composite micro-sphere material, feature
It is, the polyethylene-hydroxyapatite micrometre nanometer hierarchical structure composite microsphere diameter is 50-400 μm, and hydroxyapatite is received
Rice grain length is 20-100nm.
8. polyethylene as claimed in claim 7-hydroxyapatite micrometre 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
Cultivating system, which is stated, with polyethylene described in claim 6 or 7-hydroxyapatite micrometre nanometer hierarchical structure composite micro-sphere material is
Effective component.
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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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110172199A (en) * | 2019-06-25 | 2019-08-27 | 扬州大学 | A kind of preparation method of hydroxyapatite/ultra-high molecular weight polyethylene nano-complex |
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| CN107189295A (en) * | 2017-06-29 | 2017-09-22 | 倪群 | A kind of preparation method of hydrophobically modified hydroxyapatite polyvinyl alcohol composite material |
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2019
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Patent Citations (3)
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| JPH03254834A (en) * | 1990-03-02 | 1991-11-13 | Sangi Co Ltd | Adsorbent |
| CN104709888A (en) * | 2013-12-13 | 2015-06-17 | 中国科学院宁波材料技术与工程研究所 | Hydroxyapatite/nanometer diamond composite powder preparation method and product thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110172199A (en) * | 2019-06-25 | 2019-08-27 | 扬州大学 | A kind of preparation method of hydroxyapatite/ultra-high molecular weight polyethylene nano-complex |
| CN110172199B (en) * | 2019-06-25 | 2022-03-01 | 扬州大学 | Preparation method of hydroxyapatite/ultra-high molecular weight polyethylene nano composite |
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