CN105754934B - Dental pulp stem cell, preparation method and its related engineering material of bone tissue - Google Patents
Dental pulp stem cell, preparation method and its related engineering material of bone tissue Download PDFInfo
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- 210000005258 dental pulp stem cell Anatomy 0.000 title claims abstract description 34
- 238000002360 preparation method Methods 0.000 title claims abstract description 14
- 210000000988 bone and bone Anatomy 0.000 title abstract description 16
- 239000000463 material Substances 0.000 title abstract description 15
- 239000007788 liquid Substances 0.000 claims abstract description 34
- 210000004027 cell Anatomy 0.000 claims abstract description 31
- 239000012679 serum free medium Substances 0.000 claims abstract description 29
- 230000029087 digestion Effects 0.000 claims abstract description 17
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 claims abstract description 10
- 239000001569 carbon dioxide Substances 0.000 claims abstract description 5
- 229910002092 carbon dioxide Inorganic materials 0.000 claims abstract description 5
- 230000004927 fusion Effects 0.000 claims abstract description 5
- 230000001413 cellular effect Effects 0.000 claims abstract description 4
- 239000006228 supernatant Substances 0.000 claims abstract description 4
- 108010019160 Pancreatin Proteins 0.000 claims description 11
- 229960003957 dexamethasone Drugs 0.000 claims description 11
- UREBDLICKHMUKA-CXSFZGCWSA-N dexamethasone Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@]2(F)[C@@H]1[C@@H]1C[C@@H](C)[C@@](C(=O)CO)(O)[C@@]1(C)C[C@@H]2O UREBDLICKHMUKA-CXSFZGCWSA-N 0.000 claims description 11
- 229940055695 pancreatin Drugs 0.000 claims description 11
- 239000001963 growth medium Substances 0.000 claims description 10
- 101800003845 Neuropeptide Y Proteins 0.000 claims description 9
- 102400000064 Neuropeptide Y Human genes 0.000 claims description 9
- KFFCKOBAHMGTMW-LGQRSHAYSA-N Forsythin Chemical compound C1=C(OC)C(OC)=CC=C1[C@H]1[C@@H](CO[C@@H]2C=3C=C(OC)C(O[C@H]4[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O4)O)=CC=3)[C@@H]2CO1 KFFCKOBAHMGTMW-LGQRSHAYSA-N 0.000 claims description 6
- 108090000526 Papain Proteins 0.000 claims description 4
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- 238000009940 knitting Methods 0.000 claims 1
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- 102100024616 Platelet endothelial cell adhesion molecule Human genes 0.000 description 3
- 102100033523 Thy-1 membrane glycoprotein Human genes 0.000 description 3
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 3
- TZSMWSKOPZEMAJ-UHFFFAOYSA-N bis[(2-methoxyphenyl)methyl] carbonate Chemical compound COC1=CC=CC=C1COC(=O)OCC1=CC=CC=C1OC TZSMWSKOPZEMAJ-UHFFFAOYSA-N 0.000 description 3
- 239000001506 calcium phosphate Substances 0.000 description 3
- 230000007547 defect Effects 0.000 description 3
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- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
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- 238000011069 regeneration method Methods 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 229940078499 tricalcium phosphate Drugs 0.000 description 3
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- 102000002260 Alkaline Phosphatase Human genes 0.000 description 2
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 2
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- 210000002709 dental papilla cell Anatomy 0.000 description 2
- 210000003074 dental pulp Anatomy 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- JYGXADMDTFJGBT-VWUMJDOOSA-N hydrocortisone Chemical compound O=C1CC[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 JYGXADMDTFJGBT-VWUMJDOOSA-N 0.000 description 2
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- UZOVYGYOLBIAJR-UHFFFAOYSA-N 4-isocyanato-4'-methyldiphenylmethane Chemical compound C1=CC(C)=CC=C1CC1=CC=C(N=C=O)C=C1 UZOVYGYOLBIAJR-UHFFFAOYSA-N 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 102000029816 Collagenase Human genes 0.000 description 1
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- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
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- 102000004190 Enzymes Human genes 0.000 description 1
- 235000019738 Limestone Nutrition 0.000 description 1
- DMGNFLJBACZMRM-UHFFFAOYSA-N O[P] Chemical compound O[P] DMGNFLJBACZMRM-UHFFFAOYSA-N 0.000 description 1
- 240000008663 Persicaria orientalis Species 0.000 description 1
- 229930003268 Vitamin C Natural products 0.000 description 1
- RGCKGOZRHPZPFP-UHFFFAOYSA-N alizarin Chemical compound C1=CC=C2C(=O)C3=C(O)C(O)=CC=C3C(=O)C2=C1 RGCKGOZRHPZPFP-UHFFFAOYSA-N 0.000 description 1
- 229910052586 apatite Inorganic materials 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 210000000845 cartilage Anatomy 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
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- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 229960002424 collagenase Drugs 0.000 description 1
- 238000004040 coloring Methods 0.000 description 1
- 210000004489 deciduous teeth Anatomy 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 239000005548 dental material Substances 0.000 description 1
- 210000001968 dental pulp cell Anatomy 0.000 description 1
- 210000004268 dentin Anatomy 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 229930182470 glycoside Natural products 0.000 description 1
- 150000002338 glycosides Chemical class 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000002607 hemopoietic effect Effects 0.000 description 1
- 229960000890 hydrocortisone Drugs 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 229910052588 hydroxylapatite Inorganic materials 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 239000006028 limestone Substances 0.000 description 1
- 210000000214 mouth Anatomy 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- VSIIXMUUUJUKCM-UHFFFAOYSA-D pentacalcium;fluoride;triphosphate Chemical compound [F-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O VSIIXMUUUJUKCM-UHFFFAOYSA-D 0.000 description 1
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000001850 reproductive effect Effects 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 229960002901 sodium glycerophosphate Drugs 0.000 description 1
- REULQIKBNNDNDX-UHFFFAOYSA-M sodium;2,3-dihydroxypropyl hydrogen phosphate Chemical compound [Na+].OCC(O)COP(O)([O-])=O REULQIKBNNDNDX-UHFFFAOYSA-M 0.000 description 1
- 210000004357 third molar Anatomy 0.000 description 1
- 235000019154 vitamin C Nutrition 0.000 description 1
- 239000011718 vitamin C Substances 0.000 description 1
Classifications
-
- 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
- C12N5/0664—Dental pulp stem cells, Dental follicle stem cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
- A61L27/3834—Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3839—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by the site of application in the body
- A61L27/3843—Connective tissue
- A61L27/3865—Dental/periodontal tissues
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
- A61L27/44—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
- A61L27/46—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix with phosphorus-containing inorganic fillers
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/54—Biologically active materials, e.g. therapeutic substances
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/12—Materials or treatment for tissue regeneration for dental implants or prostheses
-
- 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
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Abstract
The invention belongs to field of pharmaceutical biology, and in particular to a kind of dental pulp stem cell, preparation method and its related engineering material of bone tissue.Dental pulp stem cell the preparation method is as follows: taking pulp tissue, tissue digestion liquid is added after shredding, 37 DEG C, 200rpm digests 10-20min, and serum free medium terminates digestion, and piping and druming discrete cellular agglomerate obtains single discrete cell, 2000rpm is centrifuged 5min, supernatant is abandoned, serum free medium cleans twice, adds serum free medium and cell is resuspended and adjusts cell density to 1 × 103A/ml, in 37 DEG C, humidity be 95% carbon dioxide incubator in cultivate, when cell grow to 80%-90% fusion when, with after separating digesting liquid vitellophag use serum free medium secondary culture.
Description
Technical field
The invention belongs to field of pharmaceutical biology, and in particular to a kind of dental pulp stem cell, preparation method and its related bone tissue
Engineering material.
Background technique
Caused defect of teeth and absence of tooth, which have become, due to various reasons endangers the main of oral cavity or even human health
Disease, disease incidence increase year by year.Presently mainly filled by means of various dental materials to cope with tooth defect disease, although
Lesion to a certain extent can be prevented to continue to develop, but not can solve root problem.For absence of tooth although solving way
There are many diameter, but most effective and the most fundamental method or the biological regeneration of tooth.
With the fast development of regeneration medicine, effective treatment for defect of teeth and absence of tooth brings hope.
There are many interactions of research and utilization Dental epithelium and Odontogenic cysts mesenchyma to have regenerated dental tissue at present.Commonly
Odontogenic cysts mesenchyma includes dental papilla cells and Dental Pulp Cells.Since dental papilla cells are clinically difficult to obtain,
Seed cell first choice currently used for tooth body and regeneration of tooth is the cell in pulp tissue source, most important of which is that dental pulp is dry
Cell.
Dental pulp stem cell is had found within 2000 for the first time, and the discovery such as Gronthos all exists in vitro deciduous teeth and wisdom tooth
Dental pulp stem cell.This be it is a kind of have high proliferation ability, height self-renewal capacity, multinomial differentiation capability cell.Have and grinds
Study carefully and successfully constructs dental pulp, dentine and dental tissue using the cell.Dental pulp stem cell (dental pulp stem
Cell, DPSC) with other tissue-derived stem cells have many similarities on biological characteristics.Gronthos etc. is to tooth
The cloning efficiency of marrow stem cell is studied, with bone marrow stroma stem cell (bone marrow stromal cell,
BMSC) after comparison, it was found that: the cloning efficiency of the DPSC in tooth source is apparently higher than the BMSC of derived from bone marrow, this is indicated that
DPSC has compared with the higher Reproductive activity of BMSC and self-renewal capacity.And dental pulp stem cell at rouge, at cartilage and at nerve
Induction aspect have research confirm dental pulp stem cell have Multidirectional Differentiation potential, for its be implanted into vivo dental pulp stem cell make by
Damage or the tissue of decline, organ improve or restore function and provide theoretical foundation.
Summary of the invention
It is an object of the present invention to provide a kind of preparation methods of dental pulp stem cell, the specific steps are as follows:
Pulp tissue is taken, tissue digestion liquid is added after shredding, 37 DEG C, 200rpm digests 10-20min, serum free medium
Digestion is terminated, piping and druming discrete cellular agglomerate obtains single discrete cell, and 2000rpm is centrifuged 5min, abandons supernatant, free serum culture
Base cleans twice, adds serum free medium and cell is resuspended and adjusts cell density to 1 × 103A/ml, in 37 DEG C, humidity
To be cultivated in 95% carbon dioxide incubator, when cell grows to 80%-90% fusion, with separating digesting liquid vitellophag
Serum free medium secondary culture is used afterwards.
In one embodiment of the invention, the tissue digestion liquid is pancreatin containing 1g/L and 0.5g/L papain
D-Hanks liquid, pH value 7.4.The tissue digestion liquid and the envelope-bulk to weight ratio of pulp tissue are 10~16ml:1g.It is described
Separating digesting liquid is the D-Hanks liquid of the pancreatin containing 1g/L, pH value 7.4.
In another embodiment of the present invention, the serum free medium is 1-10ug/L containing IGF, neuropeptide tyrosine
The MEM culture medium of 1-20ng/L, isorientin 1-20ug/L, forsythin 1-20ug/L and dexamethasone 1-20mM.Further
In ground embodiment, the serum free medium is 5ug/L containing IGF, neuropeptide tyrosine 15ng/L, isorientin 5ug/L, Fructus Forsythiae
The MEM culture medium of glycosides 8ug/L and dexamethasone 5mM.
It is a further object to provide the dental pulp stem cell of above-mentioned preparation method preparation, the dental pulp stem cell is several
Marker CD31 and CD34 specific to hemopoietic system is not expressed；And to surface marker specific to mescenchymal stem cell
CD90 and CD105 expression rate with higher.
The third object of the present invention is to provide the preparation method for loading the compound bone holder material of above-mentioned dental pulp stem cell, tool
Steps are as follows for body:
1) dental pulp stem cell suspension is prepared；
2) bone holder material is immersed in dental pulp stem cell suspension to get compound bone holder material.
In one embodiment of the invention, the bone holder material the preparation method comprises the following steps: chitosan is dissolved in 2%
In acetum, stirring be completely dissolved to chitosan, filter 2% chitosan of weight solution；Then hydroxyl is added into solution
The water slurry of apatite and bata-tricalcium phosphate is sufficiently stirred for 24 hours, is aged 1 day, filters to obtain precipitating, is existed by heat pressing forming machines
Compacting 5min makes its molding to get bone holder material under conditions of 70 DEG C and 12MPa.Further, the chitosan, hydroxyl phosphorus
The weight ratio of lime stone and bata-tricalcium phosphate is 50:15:35.
In another embodiment of the present invention, the dental pulp stem cell suspension the preparation method comprises the following steps: take pulp tissue,
Tissue digestion liquid is added after shredding, 37 DEG C, 200rpm digests 10-20min, and serum free medium terminates digestion, blows and beats discrete thin
Born of the same parents' agglomerate obtains single discrete cell, and 2000rpm is centrifuged 5min, abandons supernatant, and serum free medium cleans twice, adds nothing
Blood serum medium is resuspended cell and adjusts cell density to 1 × 103A/ml, in 37 DEG C, the carbon dioxide culture that humidity is 95%
It cultivates in case, when cell grows to 80%-90% fusion, is passed on after separating digesting liquid vitellophag with serum free medium
Culture collects dental pulp stem cell when culture is to 3 generation, is configured to 1 × 10 with serum free medium5The dental pulp stem cell of a/ml
Suspension.In further carrying out scheme, the serum free medium is 5ug/L containing IGF, neuropeptide tyrosine 15ng/L, different Polygonum orientale
The MEM culture medium of plain 5ug/L, forsythin 8ug/L and dexamethasone 5mM.The tissue digestion liquid is pancreatin containing 1g/L and 0.5g/
The D-Hanks liquid of L papain, pH value 7.4.The tissue digestion liquid and the envelope-bulk to weight ratio of pulp tissue be 10~
16ml:1g.The separating digesting liquid is the D-Hanks liquid of the pancreatin containing 1g/L, pH value 7.4.
Specific embodiment
Below by further the present invention will be described in detail.It should be pointed out that following explanation be only to the present invention claims
The technical solution of protection for example, not to any restrictions of these technical solutions.Protection scope of the present invention is with appended
Subject to the content that claims are recorded.
1 dental pulp stem cell culture of embodiment
Pulp tissue is clamped with aseptic nipper, cuts off the pulp tissue of root tip 1mm；With ophthalmology curved scissors by pulp tissue
It is cut into 1mm3, be placed in 50mL centrifuge tube, be added 13ml tissue digestion liquid (pancreatin containing 1g/L and 0.5g/L papain
D-Hanks liquid, pH value 7.4), it is sufficiently mixed uniformly, is transferred in Tempeerature-constant air shaking table after sealing, 37 DEG C, 200rpm digestion
15min ；Isometric serum free medium is added and blows and beats discrete cellular agglomerate repeatedly, is filtered by 70 μm of cell screen clothes,
2000rpm is centrifuged 3min；It is cleaned 1~2 time using PBS, precipitating serum free medium (5ug/L containing IGF, neuropeptide tyrosine
The MEM culture medium of 15ng/L, isorientin 5ug/L, forsythin 8ug/L and dexamethasone 5mM, similarly hereinafter) be resuspended, with 1 × 103
In a/ml inoculated and cultured ware, cell suspension is mixed, 37 DEG C is put in, is cultivated in the carbon dioxide incubator that humidity is 95%；24h
After change liquid and remove non-attached cell, then change liquid 1 time, when cell, which grows to 80%~90%, to be converged, separating digesting liquid within every 3 days
(the D-Hanks liquid of the pancreatin containing 1g/L, pH value 7.4) vitellophag carries out secondary culture.
Embodiment 2
According to the cultural method of embodiment 1, P1 for when, cell viability and concentration mensuration are carried out using Trypan Blue,
It is (original in each culture dish to spread into 10000 teeth that the dental pulp stem cell in culture dish specially is collected with 0.1% pancreatin digestive juice
Marrow stem cell), then PBS cleaning is dyed with 0.4% trypan blue solution, it is (flat to calculate living cells number in each culture dish
Row test is three times), and pass through flow cytomery marker CD31, CD34, CD90 and CD105.
As a result as follows:
For the cultural method of comparative example 1 with embodiment 1, difference is only that the tissue digestion liquid of comparative example 1 is pancreas containing 1.5g/L
The D-Hanks liquid of enzyme, pH value 7.4；
For the cultural method of comparative example 2 with embodiment 1, difference is only that the tissue digestion liquid of comparative example 2 is I containing 1.5g/L
The D-Hanks liquid of Collagenase Type, pH value 7.4；
For the cultural method of comparative example 3 with embodiment 1, difference is only that the serum free medium of comparative example 3 is containing IGF
The MEM culture medium of 5ug/L, neuropeptide tyrosine 15ng/L and dexamethasone 5mM
For the cultural method of comparative example 4 with embodiment 1, difference is only that the serum free medium of comparative example 4 is containing IGF
The MEM culture medium of 5ug/L, neuropeptide tyrosine 15ng/L, isorientin 13ug/L and dexamethasone 5mM
For the cultural method of comparative example 5 with embodiment 1, difference is only that the serum free medium of comparative example 5 is containing IGF
The MEM culture medium of 5ug/L, neuropeptide tyrosine 15ng/L, forsythin 13ug/L and dexamethasone 5mM
For the cultural method of comparative example 6 with embodiment 1, difference is only that the serum free medium of comparative example 5 is Chinese patent
Serum free medium disclosed in CN201510095430 specification embodiment 1: SITE100* (sigma) 10ml, ascorbic acid
300mM, PDGF 5ug, hydrocortisone 5ug, EGF3ng, b-FGF, 3ng, PTH 100ng, more than dexamethasone 10mM, IMDM
Amount
Through flow cytomery, each group stem cell shows the marker feature of dental pulp stem cell, CD31 and CD34
Expression rate be lower than 0.5%；The expression rate of CD90 and CD105 is above 98%.
The identification of 3 Osteoblast Differentiation ability of embodiment
The dental pulp stem cell for taking P3 to obtain for well-grown close to the embodiment 1 converged respectively, uses nothing after being digested with pancreatin
Blood serum medium (embodiment 1) counts after cell suspension is made.Adjusting cell density is 1 × 105The hole /mL, 2mL/ is placed in 6 orifice plates
In.Discard culture solution to when 80% fusion when cell is long, be changed to Osteogenic Induction Medium (DMEM culture solution, sodium glycero-phosphate,
Vitamin C and dexamethasone).Continuous induction, the primary induction liquid of replacement in every 3 days, induces in stopping in the 28th day, collects cell, split
Solution extracts protein determination alkaline phosphatase, draws culture based assays osteocalcin and (measures according to the method for commercial reagent box, every group of survey
Fixed 5 samples).
As a result as follows:
Embodiment 4 loads the bone holder material preparation of dental pulp stem cell
50g chitosan is dissolved in 2% acetum, stirring is completely dissolved to chitosan, filters to obtain 2% chitosan of weight
Solution；Then it is added drop-wise in the water slurry of hydroxyapatite containing 15g and 35g bata-tricalcium phosphate, is sufficiently stirred into solution
For 24 hours, it is aged 1 day, filters to obtain precipitating, compacting 5min makes its molding under conditions of 70 DEG C and 12MPa by heat pressing forming machines, i.e.,
Obtain bone holder material.
Bone holder material is put into containing 1 × 106(embodiment 1P3 generation in the serum free medium suspension of/ml dental pulp stem cell
Cell), it stands for 24 hours, then bone holder material is transferred in 6 hole culture dishes and is trained with serum free medium (embodiment 1)
It supports, was changed the liquid once every 3 days, after culture 2 weeks, pancreatin digests the cell on bone holder material, and it is living that Trypan Blue detects cell
Property, the results showed that, dental pulp stem cell well-grown (cell activity rate is 99%) on bone holder material, and it is unexpected
It is that under conditions of no osteogenic induction agent, the dental pulp stem cell on bone bracket shows apparent Osteoblast Differentiation characteristic (alizarin
Red colouring finds apparent calcium tubercle, and content of alkaline phosphatase is 101 ± 13U/g；In culture medium BGP content be 0.15 ±
0.03).
The content of present invention merely illustrates claimed some specific embodiments, one of them or more skill
Documented technical characteristic can be combined with arbitrary one or more technical solutions in art scheme, these are combined and obtain
Technical solution also in the application protection scope, the technical solution just as obtained from these are combined is disclosed in the present invention
It is specifically recorded in content the same.
Claims (2)
1. a kind of preparation method of dental pulp stem cell, the specific steps are as follows:
Pulp tissue is taken, tissue digestion liquid is added after shredding, 37 DEG C, 200rpm digests 10-20min, and serum free medium terminates
Digestion, piping and druming discrete cellular agglomerate obtain single discrete cell, and 2000rpm is centrifuged 5min, abandons supernatant, and serum free medium is clear
It washes twice, adds serum free medium and cell is resuspended and adjusts cell density to 1 × 103A/ml is in 37 DEG C, humidity
It is cultivated in 95% carbon dioxide incubator, when cell grows to 80%-90% fusion, after separating digesting liquid vitellophag
With serum free medium secondary culture；
D-Hanks liquid of the tissue digestion liquid for pancreatin containing 1g/L and 0.5g/L papain, pH value 7.4, described group
The envelope-bulk to weight ratio for knitting digestive juice and pulp tissue is 10~16ml:1g；
The separating digesting liquid is the D-Hanks liquid of the pancreatin containing 1g/L, pH value 7.4；
The serum free medium is 1-10ug/L containing IGF, neuropeptide tyrosine 1-20ng/L, isorientin 1-20ug/L, forsythin
The MEM culture medium of 1-20ug/L and dexamethasone 1-20mM.
2. the preparation method of dental pulp stem cell according to claim 1, which is characterized in that the serum free medium be containing
The MEM culture medium of IGF 5ug/L, neuropeptide tyrosine 15ng/L, isorientin 5ug/L, forsythin 8ug/L and dexamethasone 5mM.
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| CN106377799A (en) * | 2016-10-13 | 2017-02-08 | 南通大学附属医院 | Preparation method of dental pulp stem cell and chitosan scaffold complex |
| CN108753695A (en) * | 2018-06-11 | 2018-11-06 | 南京泰盛生物科技有限公司 | A kind of type I collagen subgroup and its dryness detection method by cell surface marker specific enrichment |
| CN110004114A (en) * | 2019-04-02 | 2019-07-12 | 浙江优牙生物科技有限公司 | A kind of serum free medium of dental pulp stem cell |
| CN110438069B (en) * | 2019-08-22 | 2022-11-22 | 深圳泽医细胞治疗集团有限公司 | Application of forsythiaside in promoting chondrogenic differentiation of human adipose mesenchymal stem cells in vitro |
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