CN108998412A - A method of preparing mineralising extracellular matrix-cell microballoon - Google Patents

A method of preparing mineralising extracellular matrix-cell microballoon Download PDF

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CN108998412A
CN108998412A CN201810950574.3A CN201810950574A CN108998412A CN 108998412 A CN108998412 A CN 108998412A CN 201810950574 A CN201810950574 A CN 201810950574A CN 108998412 A CN108998412 A CN 108998412A
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cell
microballoon
mineralising
extracellular matrix
follows
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刘燕
周彦恒
付翠翠
罗聃
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Hangzhou Hengrui Biotechnology Co Ltd
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Hangzhou Hengrui Biotechnology Co Ltd
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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
    • 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
    • C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/32—Amino acids
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/35—Polyols, e.g. glycerin, inositol
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/70—Undefined extracts
    • C12N2500/80—Undefined extracts from animals
    • C12N2500/84—Undefined extracts from animals from mammals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/10—Mineral substrates

Abstract

The invention discloses a kind of methods for preparing mineralising extracellular matrix-cell microballoon, it includes the following steps: the preparation of mineralized liquid, the preparation of three-dimensional microballoon, the mineralising and the material means of supplementing out economy of cell microsphere, a kind of method design cycle preparing mineralising extracellular matrix-cell microballoon is reasonable, it passes through the method for biology culture, carry out three-dimensional mineralized dentin matrix culture cell microsphere, compared with the cell of traditional external two dimension culture, the cell that the present invention is cultivated contains more cell biology information, there is the forming process of good human simulation bone tissue, a kind of method preparing mineralising extracellular matrix-cell microballoon is worth promoting and using in field of biomedicine.

Description

A method of preparing mineralising extracellular matrix-cell microballoon
Technical field
The present invention relates to field of biomedicine, the method for specifically a kind of preparation mineralising extracellular matrix-cell microballoon.
Background technique
Bone tissue is by osteoblast extracellular matrix secretion, and to Extracellular Matrix Secretion matrix by way of the slurry of class top Vesicle, contains mineral ion in vesicle, and minerals are discharged into extracellular matrix after rupturing under the action of enzyme by matrix vesicle Afterwards, it under the action of non-collagen, is deposited in collagenous fibres in the form of nanometer hydroxyapatite, forms the basic knot of bone Structure, mineralized collagen fiber, the regeneration of bone and the emphasis direction for repairing always medical research.
In recent years, the forming process of in-vitro simulated bone becomes the urgent issue to be resolved of researchers, because bone tissue is Complete three-dimensional configuration, external used cell is largely two-dimentional culture, loses many cellular informatics, not well The forming process of human simulation bone.
Aiming at the problem that being previously mentioned in background technique, the present invention is intended to provide a kind of, to prepare mineralising extracellular matrix-cell micro- The method of ball.
Summary of the invention
The purpose of the present invention is to provide a kind of methods for preparing mineralising extracellular matrix-cell microballoon, to solve above-mentioned back The problem of being proposed in scape technology.
To achieve the above object, the invention provides the following technical scheme:
A method of preparing mineralising extracellular matrix-cell microballoon comprising following steps: the preparation of mineralized liquid, three-dimensional The preparation of microballoon, the mineralising of cell microsphere and the material means of supplementing out economy, the preparation embodiment of the mineralized liquid are by 4.2mM's K2HPO4 is added complete medium and forms Mineralized Culture base A, and the poly-aspartate of the CaCl2 of 9mM and 0.2mg/ml has been added Mineralized Culture base B is formed in full culture medium；The three-dimensional microballoon preparation embodiment is that rat source medulla mesenchyma is dry thin Born of the same parents (P3) are inoculated in dimensional culture plate；The mineralising embodiment of the cell microsphere are as follows: put the microballoon in dimensional culture plate Mineralized Culture base is placed in be cultivated；The material means of supplementing out economy embodiment is using scanning electron microscope (SEM) and energy spectrum analysis The microscopic appearance and elemental composition of the different cell microspheres of instrument (EDX) observation measure different microballoons using atomic force microscope The active detection of mechanical property, microballoon inner cell, the detection of microballoon inner cell distribution and the expression analysis by osteogenesis gene The osteogenic ability of microballoon.
As a further solution of the present invention: the complete medium is configured by following ingredient: 20% cow's serum, 100U/ Ml penicillin/streptomycin, 2mM glutamine, α-MEM culture medium, 55 μM of 2ME2 and 55 μM of ground plug rice Loose sodium phosphate, the α-MEM culture medium are skilled artisans recognize that common sense culture medium；Preparing for the three-dimensional microballoon is specific Embodiment are as follows: the rat source mesenchymal stem cell (P3) being inoculated in dimensional culture plate, in 37 DEG C, 5% CO2's Apply the cell mass (CS) that complete medium culture in case obtains microballoon form afterwards for 24 hours；The mineralising of the cell microsphere is specifically real Apply mode are as follows: the Mineralized Culture base is mixed by Mineralized Culture base A and Mineralized Culture base B 1:1, in the mixed solution Ca2+Concentration is 4.5mM, PO43-Concentration is 2.1mM, the microballoon in mixed Mineralized Culture base culture tissue culture plate, for 24 hours After obtain mineralising extracellular matrix stem cell microballoon (MECS).
As a further solution of the present invention: described different with energy depressive spectroscopy (EDX) observation using scanning electron microscope (SEM) The microscopic appearance and elemental composition specific embodiment of cell microsphere are as follows: by the different cell microspheres after cultivating 5 days through 3.7% After paraformaldehyde is fixed, through gradient alcohol dehydration (50%-100%), after metal spraying, observed under 15kV voltage；It is described to use original Sub- force microscope measures the mechanical property specific embodiment of different microballoons are as follows: indoors in the measurement under conventional environment, uses Derjaguin-Muller-Toporov (DMT) models fitting power is to separation graph embedding curve, to calculate Young's modulus.Every group Select 2 samples, each samples selection 3 figures select 5 points in each figure, every group so totally 30 data, and use Anova Variance analysis.
As a further solution of the present invention: the active detection specific embodiment of microballoon inner cell are as follows: 5ul is added Into 10ml PBS, the concussion that is vortexed mixes 16mMPI storing liquid after equilibrium at room temperature, obtains the PI solution of 8uM；By 5ul 4uM's Calcium flavin AM liquid storage is added in the PI solution of 10ml, is vortexed and is mixed, it is ensured that mixes well；Acquired solution (2uM calcium flavin AM With 8uM PI), direct staining cell, to observe the activity of microballoon inner cell；The detection of the microballoon inner cell distribution is specifically real Apply mode are as follows: the cell microsphere after collection after sheep blood serum closing, is incubated for phalloidine through Triton-X rupture of membranes, and with containing 4 ', 6- diacetyl -2-phenylindone (DAPI) mountant mounting, under the microscope；It is described micro- by the expression analysis of osteogenesis gene The osteogenic ability specific embodiment of ball are as follows: after washing three times with PBS, extract by osteogenic induction and micro- without two kinds of osteogenic induction The RNA of ball calculates the expression of analysis Bone formation-related gene ALP, BMP-2 by PCR.
As a further solution of the present invention: the biology in order to verify prepared mineralising extracellular matrix-cell microballoon Functionality devises repair of cranial defects embodiment:
A kind of repair of cranial defects embodiment comprising following steps:
1) prepare material beta-TCP and two kinds of microballoons according to defect area；
2) CS, MECS and β-TCP of collection and blank control are implanted into defective region, gelfoam covering, suture respectively；
3) it is put to death after 8 weeks, micro-CT and histological examination defective region skeletonization situation；
4) with HE, Masson dyeing, immunofluorescence dyeing observation.
As a further solution of the present invention: described to be implanted into CS, MECS and β-TCP of collection and blank control respectively Defective region, gelfoam covering, the specific embodiment of suture are as follows: 6-8 week old SD rat, the intraperitoneal injection of 1% yellow Jackets Anesthesia, cropping is fixed, sterile drape；In cranium top row t shaped incision, fascia, exposure skull bone face are separated；Use planting machine (W& H) 1200 turns/min cooperates annular bone drill (outer diameter 4.8mm) to abrade holostrome bone tissue, sterile physiological respectively in calvarium sutura two sides Brine-cooled, hemostasis by compression；CS, MECS, β-TCP and blank control are taken, defect, skin suture are implanted into.
As a further solution of the present invention: the micro-CT and histological examination defective region skeletonization situation is specifically real Apply mode are as follows: after 8w, sample is put to death, it is fixed, and Skyscan 1174 is used, (Bruker) scanning defect area (voltage 53kV, Electric current 810A), rebuild using NRecon, CTAn and CTvox (Bruker) software and be analyzed with data, calculates defective region Bone volume, each sample repeat to survey three times, carry out variance analysis to data using SPSS；Described is dyed with HE, Masson, is exempted from Epidemic disease Fluorescent Staining Observation specific embodiment are as follows: take defective region to organize, decalcification 3-4 weeks in the EDTA decalcifying Fluid containing sucrose；Ladder Degree dehydration of alcohol, waxdip, paraffin embedding, hard tissue slicing machine-cut piece, thickness 5um, dimethylbenzene-alcohol dewax to water, hematoxylin- Yihong (HE) dyeing or Masson dyeing, mounting, microscopically observation.
Compared with prior art, the beneficial effects of the present invention are: described a kind of to prepare mineralising extracellular matrix-cell micro- The method design cycle of ball is reasonable, by the method for biology culture, three-dimensional mineralized dentin matrix culture cell microsphere is carried out, with biography The cell of the external two dimension culture of system is compared, and the cell that the present invention is cultivated contains more cell biology information, has very The forming process of good human simulation bone tissue, a kind of method preparing mineralising extracellular matrix-cell microballoon are worth Field of biomedicine is promoted and is used.
Detailed description of the invention
Fig. 1 is the shape of the cell mass (CS) and mineralising extracellular matrix stem cell microballoon (MECS) after culture in 24 hours State structural schematic diagram.
Fig. 2 is microscopic appearance structural schematic diagram of the different cell microspheres at scanning electron microscope (SEM).
Fig. 3 is that elemental composition of the different cell microspheres under energy depressive spectroscopy (EDX) analysis forms result schematic diagram.
Fig. 4 is the bar shaped statistical chart of the Young's modulus of CS and the Young's modulus of MECS.
Fig. 5 is the dye distribution result schematic diagram of the active detection of microballoon inner cell.
Fig. 6 is the dye distribution result schematic diagram of microballoon inner cell distribution.
Fig. 7 is the bar shaped statistical chart of the expression analysis of osteogenesis gene.
Fig. 8 is animal model schematic diagram.
Fig. 9 is the result schematic diagram that micro-CT checks defective region skeletonization situation.
Figure 10 is the result schematic diagram of histological examination defective region skeletonization situation.
Figure 11 is the result schematic diagram that defective tissue is dyed with HE, Masson.
Figure 12 is the result schematic diagram with immunofluorescence dyeing defective tissue.
Specific embodiment
Technical solution of the present invention is described in more detail With reference to embodiment.
Fig. 1-12 is please referred to, a method of preparing mineralising extracellular matrix-cell microballoon comprising following steps: mineralising The preparation of liquid, the preparation of three-dimensional microballoon, cell microsphere mineralising and the material means of supplementing out economy, the preparation embodiment party of the mineralized liquid Formula is that complete medium is added in the K2HPO4 of 4.2mM to form Mineralized Culture base A, by the poly- day of the CaCl2 of 9mM and 0.2mg/ml Aspartic acid, which is added in complete medium, forms Mineralized Culture base B；The three-dimensional microballoon preparation embodiment is by rat source bone Bone marrow-drived mesenchymal stem (P3) is inoculated in dimensional culture plate；The mineralising embodiment of the cell microsphere are as follows: by dimensional culture Microballoon in plate is placed in Mineralized Culture base and is cultivated；The material means of supplementing out economy embodiment is using scanning electron microscope (SEM) microscopic appearance of cell microspheres different with energy depressive spectroscopy (EDX) observation and elemental composition, using atomic force microscope Measure the active detection of the mechanical properties of different microballoons, microballoon inner cell, the distribution of microballoon inner cell detection and pass through skeletonization The osteogenic ability of the expression analysis microballoon of gene.
The complete medium is configured by following ingredient: 20% cow's serum, 100U/ml penicillin/streptomycin, 2mM paddy ammonia Amide, α-MEM culture medium, 55 μM of 2ME2 and 55 μM of dexamethasone sodium phosphate, the α-MEM culture medium For skilled artisans recognize that common sense culture medium.
The preparation specific embodiment of the three-dimensional microballoon are as follows: filled between the rat source marrow being inoculated in dimensional culture plate Matter stem cell (P3), in 37 DEG C, 5% CO2Deposited case in complete medium culture obtain the cell mass of microballoon form afterwards for 24 hours (CS)。
The mineralising specific embodiment of the cell microsphere are as follows: the Mineralized Culture base is trained by Mineralized Culture base A and mineralising It supports base B 1:1 to be mixed, Ca in the mixed solution2+Concentration is 4.5mM, PO43-Concentration is 2.1mM, mixed mineralising Microballoon in culture medium culture tissue culture plate obtains mineralising extracellular matrix stem cell microballoon (MECS) afterwards for 24 hours.
The cell mass (CS) and mineralising extracellular matrix stem cell microballoon (MECS) obtained after culture in 24 hours exists Under electron microscope structures observation, their morphosis is as shown in Figure 1, observation result are as follows: is formed in agarose culture plate big Small uniform, the similar cell microsphere of form, CS and MECS have larger difference in appearance, and CS diameter is less than MECS, and CS diameter is 113.3 ± 20.8um, and MECS diameter is 246.7 ± 15.3um, almost fills out entire micropore.
The microscopic appearance using scanning electron microscope (SEM) different with energy depressive spectroscopy (EDX) observation cell microsphere and Elemental composition specific embodiment are as follows: by the different cell microspheres after cultivating 5 days after 3.7% paraformaldehyde is fixed, through gradient Dehydration of alcohol (50%-100%) after metal spraying, is observed under 15kV voltage, and observation result is as shown in Figures 2 and 3, it was therefore concluded that Are as follows: CS shows the smooth pattern in surface, is cellular morphology, and shows there is many around cell (red arrow) after MECS amplification The little particle (black arrow) of nanometer (nm) rank, similar hydroxyapatite, shows after amplification, small particle diameters 376.9 ± 59.2nm is dispersed in distribution and is surrounded in the extracellular matrix (ECM) of cell peripheral；CS regional part is made of C, O element substantially, No other extra elements exist, it was demonstrated that and it is cell component in CS, and in MECS other than C, O, there is also Ca, P element, according to The elemental composition in surveyed region judges it for hydroxyapatite.
The mechanical property specific embodiment that different microballoons are measured using atomic force microscope are as follows: conventional ring indoors In measurement under border, using Derjaguin-Muller-Toporov (DMT) models fitting power to separation graph embedding curve, come Calculate Young's modulus.2 samples of every group of selection, each samples selection 3 figures select 5 points in each figure, and every group so totally 30 Data, and use Anova variance analysis；Experimental data according to Fig. 4 experimental result as shown in figure 4, draw a conclusion are as follows: CS's Young's modulus is 265 ± 33MPa, and the Young's modulus of MECS is 1428 ± 78MPa, and the Young's modulus of MECS is significantly higher than CS, says Bright nanometer hydroxyapatite substantially improves the hardness and tenability of MECS, to improve the mechanical mechanics of MECS Energy.
The active detection specific embodiment of microballoon inner cell are as follows: the 16mMPI storage after 5ul equilibrium at room temperature is added Into 10ml PBS, the concussion that is vortexed mixes liquid, obtains the PI solution of 8uM；The calcium flavin AM liquid storage of 5ul 4uM is added to 10ml PI solution in, be vortexed mix, it is ensured that mix well；Acquired solution (2uM calcium flavin AM and 8uM PI), direct staining is thin Born of the same parents, to observe the activity of microballoon inner cell；Experimental result as shown in figure 5, green have a finger in every pie of born of the same parents shows living cells, show extremely carefully by red have a finger in every pie of cell Born of the same parents.Position has a large amount of red dye to show among CS, is shown as dead cell, and does not find a large amount of erythrophils in MECS, dead cell Account for the percentage statistics such as Fig. 5, P < 0.01 of sum.
The detection specific embodiment of the microballoon inner cell distribution are as follows: the cell microsphere after collection, it is broken through Triton-X Film after sheep blood serum closing, is incubated for phalloidine, and sealed with containing 4 ', 6- diacetyl -2-phenylindone (DAPI) mountant Piece, under the microscope；Result is observed as shown in fig. 6, drawing a conclusion according to Fig. 6 are as follows: in Single spheroid in CS and MECS Nucleus is uniformly distributed, and forms the ball with three-D space structure, and the F-actin of MECS understands compared with CS Subfilament Structure, and arrangement causes Close, expression intensity is high, illustrates that the presence of nanometer hydroxyapatite has conducive to the formation of the cytoskeleton in MECS extracellular matrix And good state is kept, by DAPI as it can be seen that the density of nucleus is greater than the density of CS nucleus in MECS, in two kinds of microballoons Nucleus be all uniformly distributed, there is no partial region is crowded or the case where be unevenly distributed.
The osteogenic ability specific embodiment of the expression analysis microballoon by osteogenesis gene are as follows: after washing three times with PBS, Extract pass through osteogenic induction and the RNA without two kinds of microballoons of osteogenic induction, by PCR calculate analysis Bone formation-related gene ALP, The expression of BMP-2, experimental result is as shown in fig. 7, according to Fig. 7, it was therefore concluded that: the Bone formation-related gene ALP of MECS and The expression quantity of BMP-2 is all higher than CS group in osteogenic induction and non-induced group in 5d and 10d, and the result shows nano-hydroxy-apatites The expression quantity that the presence of stone promotes Bone formation-related gene in cell RNA in MECS increases, to improve the skeletonization energy of MECS Power.
In order to verify the Biological Functional of prepared mineralising extracellular matrix-cell microballoon, devises skull defeci and repair Multiple embodiment:
Fig. 8-11 is please referred to, a kind of repair of cranial defects embodiment comprising following steps:
1) prepare material beta-TCP and two kinds of microballoons according to defect area；
2) CS, MECS and β-TCP of collection and blank control are implanted into defective region, gelfoam covering, suture respectively；
3) it is put to death after 8 weeks, micro-CT and histological examination defective region skeletonization situation；
4) with HE, Masson dyeing, immunofluorescence dyeing observation；
Described is implanted into CS, MECS and β-TCP of collection and blank control defective region, gelfoam covering, seam respectively The specific embodiment of conjunction are as follows: 6-8 week old SD rat, 1% yellow Jackets intraperitoneal injection of anesthesia, cropping is fixed, disinfection paving Towel；In cranium top row t shaped incision, fascia, exposure skull bone face are separated；Cooperate annular using planting machine (W&H) 1200 turns/min Bone drill (outer diameter 4.8mm) abrades holostrome bone tissue in calvarium sutura two sides respectively, and sterile saline is cooling, hemostasis by compression；It takes CS, MECS, β-TCP and blank control are implanted into defect, skin suture；Experimental phenomena is as shown in Figure 8.
The micro-CT and histological examination defective region skeletonization situation specific embodiment are as follows: after 8w, at sample Extremely, fixed, and use Skyscan 1174, (Bruker) scanning defect area (voltage 53kV, electric current 810A), using NRecon, CTAn and CTvox (Bruker) software rebuild and is analyzed with data, calculates defective region bone volume, and each sample repeats to survey Three times, variance analysis is carried out to data using SPSS, obtained experimental data result is as shown in Figure 9, it was therefore concluded that are as follows: after 8w The comparison of micro-CT newborn bone amount: Control=1.71 ± 0.04mm3, β-TCP=6.33 ± 0.55mm3, CS=8.05 ± There are apparent difference between 0.08mm3, MECS=12.01 ± 0.21mm3 group, after 8w, MECS group defect area is largely repaired It is multiple, hence it is evident that be better than other groups.
Described observes specific embodiment with HE, Masson dyeing, immunofluorescence dyeing are as follows: and take defective region to organize, in Decalcification 3-4 weeks in EDTA decalcifying Fluid containing sucrose；Gradient alcohol dehydration, waxdip, paraffin embedding, hard tissue slicing machine-cut piece are thick 5um is spent, dimethylbenzene-alcohol dewaxes to water, hematoxylin-eosin (HE) dyeing or Masson dyeing, mounting, microscopically observation； Acquired results are as shown in Figure 10 and Figure 11, it was therefore concluded that: MECS group visible material is degraded completely substantially, and area of new bone is filled in entirely Also there are a large amount of mature New born formations in defect area, center；The visible Bone formation-related gene in MECS group of immunofluorescence dyeing BMP-2 is above other groups at the expression quantity of blood vessel related gene CD31.
Better embodiment of the invention is explained in detail above, but the present invention is not limited to above-mentioned embodiment party Formula within the knowledge of one of ordinary skill in the art can also be without departing from the purpose of the present invention Various changes can be made.

Claims (9)

1. a kind of method for preparing mineralising extracellular matrix-cell microballoon comprising following steps: the preparation of mineralized liquid, three-dimensional are micro- The preparation of ball, the mineralising of cell microsphere and the material means of supplementing out economy, it is characterised in that: the preparation embodiment of the mineralized liquid is Complete medium is added in the K2HPO4 of 4.2mM and forms Mineralized Culture base A, by the poly- asparagus fern ammonia of the CaCl2 of 9mM and 0.2mg/ml Acid, which is added in complete medium, forms Mineralized Culture base B；The three-dimensional microballoon preparation embodiment is will be between the marrow of rat source Mesenchymal stem cells (P3) are inoculated in dimensional culture plate；The mineralising embodiment of the cell microsphere are as follows: will be in dimensional culture plate Microballoon be placed in Mineralized Culture base and cultivated；The material means of supplementing out economy embodiment be using scanning electron microscope (SEM) and The microscopic appearance and elemental composition of the different cell microspheres of energy depressive spectroscopy (EDX) observation are measured not using atomic force microscope With the active detection of the mechanical property of microballoon, microballoon inner cell, the distribution of microballoon inner cell detection and pass through osteogenesis gene The osteogenic ability of expression analysis microballoon.
2. a kind of method for preparing mineralising extracellular matrix-cell microballoon according to claim 1, it is characterised in that: described Complete medium is configured by following ingredient: 20% cow's serum, 100U/ml penicillin/streptomycin, 2mM glutamine, α-MEM training Base, 55 μM of 2ME2 and 55 μM of dexamethasone sodium phosphate are supported, the α-MEM culture medium is art technology Personnel generally acknowledge common sense culture medium.
3. a kind of method for preparing mineralising extracellular matrix-cell microballoon according to claim 1, it is characterised in that: described The preparation specific embodiment of three-dimensional microballoon are as follows: the rat source mesenchymal stem cell being inoculated in dimensional culture plate (P3), in 37 DEG C, CO that concentration is 5%2Deposited case in complete medium culture obtain the cell mass of microballoon form afterwards for 24 hours (CS)；The mineralising specific embodiment of the cell microsphere are as follows: the Mineralized Culture base is by Mineralized Culture base A and Mineralized Culture base B 1:1 is mixed, Ca in the mixed solution2+Concentration is 4.5mM, PO43-Concentration is 2.1mM, mixed Mineralized Culture Microballoon in base culture tissue culture plate obtains mineralising extracellular matrix stem cell microballoon (MECS) afterwards for 24 hours；It is described to use scanning The microscopic appearance and elemental composition specific embodiment of Electronic Speculum (SEM) cell microsphere different with energy depressive spectroscopy (EDX) observation Are as follows: by the different cell microspheres after cultivating 5 days after 3.7% paraformaldehyde is fixed, through gradient alcohol dehydration (50%- 100%) it, after metal spraying, is observed under 15kV voltage；The mechanical property for measuring different microballoons using atomic force microscope is specific Embodiment are as follows: indoors in the measurement under conventional environment, using Derjaguin-Muller-Toporov (DMT) models fitting Power is to separation graph embedding curve, to calculate Young's modulus, 2 samples of every group of selection, each samples selection 3 figures, Mei Getu In select 5 points, every group so totally 30 data, and use Anova variance analysis.
4. a kind of method for preparing mineralising extracellular matrix-cell microballoon according to claim 1, it is characterised in that: described The active detection specific embodiment of microballoon inner cell are as follows: the 16mMPI storing liquid after 5ul equilibrium at room temperature is added is to 10ml PBS In, the concussion that is vortexed mixes, and obtains the PI solution of 8uM；The calcium flavin AM liquid storage of 5ul 4uM is added in the PI solution of 10ml, It is vortexed and mixes, it is ensured that mix well, acquired solution (2uM calcium flavin AM and 8uM PI), direct staining cell, observe in microballoon The activity of cell；The detection specific embodiment of the microballoon inner cell distribution are as follows: the cell microsphere after collection, through Triton-X Rupture of membranes after sheep blood serum closing, is incubated for phalloidine, and sealed with containing 4 ', 6- diacetyl -2-phenylindone (DAPI) mountant Piece, under the microscope.
5. a kind of method for preparing mineralising extracellular matrix-cell microballoon according to claim 1, it is characterised in that: described Pass through the osteogenic ability specific embodiment of the expression analysis microballoon of osteogenesis gene are as follows: after washing three times with PBS, extract and pass through skeletonization Induction and the RNA without two kinds of microballoons of osteogenic induction calculate the expression feelings of analysis Bone formation-related gene ALP, BMP-2 by PCR Condition.
6. a kind of method for preparing mineralising extracellular matrix-cell microballoon described in -5 according to claim 1, in order to verify the party The Biological Functional of mineralising extracellular matrix-cell microballoon, devises repair of cranial defects embodiment obtained by method, The following steps are included:
1) prepare material beta-TCP and two kinds of microballoons according to defect area；
2) CS, MECS and β-TCP of collection and blank control are implanted into defective region, gelfoam covering, suture respectively；
3) it is put to death after 8 weeks, micro-CT and histological examination defective region skeletonization situation；
4) with HE, Masson dyeing, immunofluorescence dyeing observation.
7. repair of cranial defects embodiment according to claim 6, it is characterised in that: it is described by the CS of collection, MECS and β-TCP and blank control are implanted into defective region, gelfoam covering, the specific embodiment of suture are as follows: 6-8 week old respectively SD rat, 1% yellow Jackets intraperitoneal injection of anesthesia, cropping is fixed, sterile drape；In cranium top row t shaped incision, muscle is separated Film, exposure skull bone face；Cooperate annular bone drill (outer diameter 4.8mm) in calvarium sutura two using planting machine (W&H) 1200 turns/min Side abrades holostrome bone tissue respectively, and sterile saline is cooling, hemostasis by compression；CS, MECS, β-TCP and blank control are taken, is implanted into Defect, skin suture.
8. repair of cranial defects embodiment according to claim 6, it is characterised in that: the micro-CT and tissue It learns and checks defective region skeletonization situation specific embodiment are as follows: after 8w, sample is put to death, it is fixed, and with Skyscan 1174, (Bruker) defect area (voltage 53kV, electric current 810A) is scanned, uses NRecon, CTAn and CTvox (Bruker) software Rebuild and analyzed with data, calculate defective region bone volume, each sample repeats to survey three times, carries out variance to data using SPSS Analysis.
9. repair of cranial defects embodiment according to claim 6, it is characterised in that: described is contaminated with HE, Masson Color, immunofluorescence dyeing observe specific embodiment are as follows: take defective region to organize, the decalcification 3-4 in the EDTA decalcifying Fluid containing sucrose Week；Gradient alcohol dehydration, waxdip, paraffin embedding, hard tissue slicing machine-cut piece, thickness 5um, dimethylbenzene-alcohol dewax to water, Soviet Union H & E (HE) dyeing or Masson dyeing, mounting, microscopically observation.
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Application publication date: 20181214