CN109749119A - Polylactic acid-glycolic base apatite micron/nano multilevel structure composite microsphere material and application - Google Patents
Polylactic acid-glycolic base apatite micron/nano multilevel structure composite microsphere material and application Download PDFInfo
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- CN109749119A CN109749119A CN201910097397.3A CN201910097397A CN109749119A CN 109749119 A CN109749119 A CN 109749119A CN 201910097397 A CN201910097397 A CN 201910097397A CN 109749119 A CN109749119 A CN 109749119A
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Abstract
The invention discloses a kind of polylactic acid-glycolic base apatite micron/nano multilevel structure composite microsphere material and application, polylactic acid-glycolic base apatite micron/nano multilevel structure composite microsphere material of the invention is with polylactic acid and CaCO3It is scattered in methylene chloride, the first solution is obtained, using microflow control technique, the first solution is instilled in polyvinyl alcohol water solution, by control PLA concentration, the drop of polylactic acid speed, polyvinyl alcohol water solution concentration and mixing speed, uniform polylactic acid-CaCO is obtained3Microballoon.Microballoon is immersed into K2HPO4CaCO in solution, with microsphere surface3It is reacted under water bath with thermostatic control, after deionized water and washes of absolute alcohol, vacuum drying obtains the polylactic acid composite microspheres of pan coating hydroxyapatite nano-structure.The micro-sphere structure stabilization, size uniformity, diameter are 50-400 μm.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 polylactic acid-glycolic base apatite micron/nano
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.
Microballoon is mainly used for the fields such as medicament slow release, cytoskeleton and cell infusion at present, does not have also thin at soma using microballoon promotion
Born of the same parents are proliferated and maintain the report of adult stem cell stemness.
Summary of the invention
For the above-mentioned prior art, it is multistage that the object of the present invention is to provide a kind of polylactic acid-glycolic base apatite micron/nanos
Structure composite micro-sphere material.Polylactic acid-glycolic base apatite micron/nano multilevel structure composite microsphere material of the invention has good
Good cell compatibility, can promote adult stem cell proliferation, cell survival rate can maintain adult stem cell 98% or more
Stemness, using the adult after polylactic acid-glycolic base apatite micron/nano multilevel structure composite microsphere material amplification of the invention
Stem cell 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 polylactic acid-glycolic base apatite micron/nano multilevel structure complex microsphere material
The preparation method of material, comprising the following steps:
(1) by polylactic acid and dissolution of calcium carbonate in methylene chloride, the first solution is made；
(2) the first solution is instilled in polyvinyl alcohol water solution using microflow control technique, is included under agitation
The polylactic acid microsphere of calcium carbonate；
(3) polylactic acid microsphere calciferous will be wrapped and immerse K2HPO4In solution, 3-7 is reacted under 20-30 DEG C of water bath with thermostatic control
It, harvests microballoon, washs, dry, that is, polylactic acid-glycolic base apatite micron/nano multilevel structure complex microsphere material is prepared
Material.
In step (1), in first solution, the weight percentage of polylactic acid is 1-10%, the molecular weight of polylactic acid
It is 50,000-50 ten thousand, the weight percentage of calcium carbonate is 1-10%.
It is described to be instilled the first solution in polyvinyl alcohol water solution using microflow control technique in step (2), specifically: it is micro-
The aperture in channel is 40-400 μm, and instillation speed is 0.01mL/min-3mL/min.
In step (2), the concentration of the polyvinyl alcohol water solution is 1mg/mL-10mg/mL.
In step (2), the speed of stirring is 100rpm-1000rpm.
In step (3), the K2HPO4The concentration of solution is 0.01mol/L-0.25mol/L, pH value 6.8-7.2.
The second aspect of the present invention provides the polylactic acid-glycolic base apatite micron/nano multilevel structure of above method preparation
Composite microsphere material.The diameter of the polylactic acid-glycolic base apatite micron/nano multilevel structure composite microsphere material is 50-400 μ
m。
The third aspect of the present invention provides above-mentioned polylactic acid-glycolic base apatite micron/nano multilevel structure complex microsphere 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 cultivating system is with above-mentioned polylactic acid-glycolic base apatite micron/nano multilevel structure composite microsphere material
For 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) polylactic acid-glycolic base apatite micron/nano multilevel structure composite microsphere material of the invention, 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) polylactic acid-glycolic base apatite micron/nano multilevel structure composite microsphere material of the invention has good thin
Cell phase capacitive can promote adult stem cell proliferation, and cell survival rate can maintain the dry of adult stem cell 98% or more
Property, using thin at soma after polylactic acid-glycolic base apatite micron/nano multilevel structure composite microsphere material amplification of the invention
Born of the same parents have good differentiation potential, can be divided into skeletonization, fat and cartilage cell.
Detailed description of the invention:
Fig. 1: the preparation flow of polylactic acid-glycolic base apatite micron/nano multilevel structure composite microsphere material of the invention
Figure.
Fig. 2: the structural schematic diagram of polylactic acid-glycolic base apatite micron/nano complex microsphere of the invention.
Fig. 3: CCK8 dyeing display is grown on polylactic acid-glycolic base apatite micron/nano multilevel structure composite microsphere material
Fat mesenchymal stem cell proliferative conditions；Wherein, normal group is the cell being grown in culture dish；Material group 1-3 difference
For the cell for being grown in the polylactic acid-glycolic base apatite micron/nano multilevel structure complex microsphere that embodiment 1-3 is obtained.
Fig. 4: the AM-PI dyeing after cultivating fat mesenchymal stem cell 7 days on a different material；Wherein, it makes a living for normal group
Grow the cell in culture dish；Material group is the polylactic acid-glycolic base apatite micron/nano multistage knot for being grown in the acquisition of embodiment 3
The cell of structure complex microsphere.Green is that calcium flavin dyes, and red is that PI is dyed.
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
The polylactic acid-glycolic base apatite micron/nano multilevel structure composite microsphere 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, it is compound to give polylactic acid-glycolic base apatite micron/nano multilevel structure
The preparation method (as shown in Figure 1) of micro-sphere material, comprising the following steps:
It is basic unit with polylactic acid microsphere, in its surface synthesizing hydroxylapatite nanostructure；Specifically:
By polylactic acid and CaCO3It is dissolved in methylene chloride, using microflow control technique, it is water-soluble that solution is instilled into polyvinyl alcohol
In liquid, obtaining in stirring condition includes CaCO3Polylactic acid microsphere, microballoon is immersed into K2HPO4In solution, in 20-30 DEG C of constant temperature
It reacts 3-7 days under water-bath, is cleaned three times with deionized water, then with after washes of absolute alcohol, vacuum drying obtains pan coating hydroxyl
The polylactic acid microsphere (as shown in Figure 2) of base apatite nanostructure.
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- cream
Hydroxyapatite is grown on acid gel.
Hydroxyapatite nano-structure is grown on polylactic acid microsphere surface to realize, the present invention is by polylactic acid and calcium carbonate
It is dissolved in methylene chloride, forms the first solution；Then the first solution is instilled by polyvinyl alcohol water solution using microflow control technique
In, it obtains wrap polylactic acid microsphere calciferous under agitation；Then polylactic acid microsphere calciferous will be wrapped to immerse
K2HPO4It in solution, is reacted 3-7 days under 20-30 DEG C of water bath with thermostatic control, the calcium carbonate meeting on surface and K2HPO4Reaction forms covering
Hydroxyapatite on polylactic acid surface.
The reaction condition of above-mentioned whole preparation process is mild, and polylactic acid microsphere does not pass through high temperature melting processing, is still real
The microballoon of the heart.In addition, making the compound micro- of preparation by the concentration of polylactic acid in the first solution of control and using microflow control technique
Ball material uniform particle diameter, form are controllable.
Experiment proves that using the polylactic acid-glycolic base phosphorus with specific structure composition and pattern of the method for the present invention preparation
Lime stone micron/nano multilevel structure composite microsphere material can promote the proliferation of adult stem cell, and maintain the stemness of stem 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: the preparation of polylactic acid-glycolic base apatite micron/nano multilevel structure composite microsphere material
By 10g polylactic acid (molecular weight is 100,000) and 5gCaCO3It is dissolved in 100ml methylene chloride, using micro-fluidic skill
Art (microchannel is 400 μm), solution is instilled in polyvinyl alcohol water solution, instillation speed is 0.5mL/min；In stirring condition
It includes CaCO that (mixing speed 500rpm), which is obtained,3Polylactic acid microsphere.
It will include CaCO3Polylactic acid microsphere immerse K2HPO4In solution (concentration 0.1mol/L, pH value 7.0), in 25
It is reacted 5 days under DEG C water bath with thermostatic control, harvests microballoon, clean three times with deionized water, then with after washes of absolute alcohol, be dried in vacuo,
Polylactic acid-glycolic base apatite micron/nano multilevel structure composite microsphere material is prepared.
Embodiment 2: the preparation of polylactic acid-glycolic base apatite micron/nano multilevel structure composite microsphere material
By 5g polylactic acid (molecular weight is 500,000) and 1gCaCO3It is dissolved in 100ml methylene chloride, using microflow control technique
(microchannel is 600 μm), solution is instilled in polyvinyl alcohol water solution, and instillation speed is 3mL/min；In stirring condition (stirring
Speed is 1000rpm) it obtains comprising CaCO3Polylactic acid microsphere.
It will include CaCO3Polylactic acid microsphere immerse K2HPO4In solution (concentration 0.05mol/L, pH value 6.8), in
It is reacted 7 days under 20 DEG C of waters bath with thermostatic control, harvests microballoon, cleaned three times with deionized water, then with after washes of absolute alcohol, vacuum is dry
It is dry, that is, polylactic acid-glycolic base apatite micron/nano multilevel structure composite microsphere material is prepared.
Embodiment 3: the preparation of polylactic acid-glycolic base apatite micron/nano multilevel structure composite microsphere material
By 5g polylactic acid (molecular weight is 300,000) and 10gCaCO3It is dissolved in 100ml methylene chloride, using micro-fluidic skill
Art (microchannel is 800 μm), solution is instilled in polyvinyl alcohol water solution, instillation speed is 0.5mL/min；In stirring condition
It includes CaCO that (mixing speed 100rpm), which is obtained,3Polylactic acid microsphere.
It will include CaCO3Polylactic acid microsphere immerse K2HPO4In solution (concentration 0.25mol/L, pH value 7.2), in
It is reacted 3 days under 30 DEG C of waters bath with thermostatic control, harvests microballoon, cleaned three times with deionized water, then with after washes of absolute alcohol, vacuum is dry
It is dry, that is, polylactic acid-glycolic base apatite micron/nano multilevel structure composite microsphere material is prepared.
Test example 1:
It is micro- that source of people fat mesenchymal stem cell is seeded in polylactic acid-glycolic base apatite prepared by embodiment 1- embodiment 3
On rice nanometer hierarchical structure composite micro-sphere material, after cultivating 1,3,5 day respectively, using CCK8 staining kit, detects cell and increase
Rate is grown, is found compared with control material (culture dish), the cell proliferation rate grown on microballoon significantly increases, and with culture
The extension of time, microballoon promote the effect of cell growth more significant, illustrate that material has good biocompatibility (Fig. 3)；
It is that (dyeing is green to calcium flavin stained positive, is represented that calcium flavin-PI cell, which dyes be shown in the cell grown on microballoon anyway,
Living cells), and few cell is PI stained positive (dyeing is red, represents dead cell) (Fig. 4), is shown in polylactic acid-glycolic base
The cell grown on apatite micron/nano multilevel structure composite microsphere material keeps good bioactivity, and cell survival rate is high
Up to 98% or more.Under the action of skeletonization and adipogenic induction liquid, the fat mesenchymal stem cell harvested on microballoon can be divided into
Skeletonization and fat cell.
The result shows that polylactic acid-glycolic base apatite micron/nano multilevel structure composite microsphere material energy prepared by the present invention
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 preparation method of polylactic acid-glycolic base apatite micron/nano multilevel structure composite microsphere material, which is characterized in that
The following steps are included:
(1) by polylactic acid and dissolution of calcium carbonate in methylene chloride, the first solution is made；
(2) the first solution is instilled in polyvinyl alcohol water solution using microflow control technique, obtaining under agitation includes carbonic acid
The polylactic acid microsphere of calcium；
(3) polylactic acid microsphere calciferous will be wrapped and immerse K2HPO4In solution, reacted 3-7 days under 20-30 DEG C of water bath with thermostatic control,
Microballoon is harvested, is washed, it is dry, that is, polylactic acid-glycolic base apatite micron/nano multilevel structure composite microsphere material is prepared.
2. preparation method according to claim 1, which is characterized in that in step (1), in first solution, polylactic acid
Weight percentage be 1-10%, the molecular weight of polylactic acid is 50,000-50 ten thousand, and the weight percentage of calcium carbonate is 1-10%.
3. preparation method according to claim 1, which is characterized in that in step (2), it is described using microflow control technique by the
One solution instills in polyvinyl alcohol water solution, specifically: the aperture of microchannel is 400-800 μm, and instillation speed is 0.01mL/
min-3mL/min。
4. preparation method according to claim 1, which is characterized in that in step (2), the polyvinyl alcohol water solution it is dense
Degree is 1mg/mL-10mg/mL.
5. preparation method according to claim 1, which is characterized in that in step (2), the speed of stirring is 100rpm-
1000rpm。
6. preparation method according to claim 1, which is characterized in that in step (3), the K2HPO4The concentration of solution is
0.01mol/L-0.25mol/L, pH value 6.8-7.2.
7. the polylactic acid-glycolic base apatite micron/nano multilevel structure of method preparation described in any one of claims 1-6 is compound
Micro-sphere material.
8. polylactic acid-glycolic base apatite micron/nano multilevel structure composite microsphere material as claimed in claim 7 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 to have that cultivating system, which is stated, with polylactic acid-glycolic base apatite micron/nano multilevel structure composite microsphere material as claimed in claim 7
Imitate 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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| CN111417459A (en) * | 2019-07-05 | 2020-07-14 | 广州中大医疗器械有限公司 | Novel drug-loaded microsphere and preparation method thereof |
| CN115058389A (en) * | 2022-07-01 | 2022-09-16 | 济南大学 | Chlorogenic acid-calcium nano sustained-release agent and application thereof in promoting osteogenic differentiation of stem cells |
| CN116236443A (en) * | 2023-05-12 | 2023-06-09 | 北京青藤谷禧干细胞科技研究院有限公司 | Mesenchymal stem cell repairing injection and preparation method thereof |
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