CN105754934A - Dental pulp stem cell, preparation method thereof and related bone tissue engineering material - Google Patents
Dental pulp stem cell, preparation method thereof and related bone tissue engineering material Download PDFInfo
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- CN105754934A CN105754934A CN201610127648.4A CN201610127648A CN105754934A CN 105754934 A CN105754934 A CN 105754934A CN 201610127648 A CN201610127648 A CN 201610127648A CN 105754934 A CN105754934 A CN 105754934A
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- 210000005258 dental pulp stem cell Anatomy 0.000 title claims abstract description 42
- 210000000988 bone and bone Anatomy 0.000 title claims abstract description 21
- 239000000463 material Substances 0.000 title claims abstract description 20
- 238000002360 preparation method Methods 0.000 title claims abstract description 19
- 239000007788 liquid Substances 0.000 claims abstract description 38
- 210000004027 cell Anatomy 0.000 claims abstract description 33
- 239000012679 serum free medium Substances 0.000 claims abstract description 30
- 210000001519 tissue Anatomy 0.000 claims abstract description 28
- 230000029087 digestion Effects 0.000 claims abstract description 18
- 239000006228 supernatant Substances 0.000 claims abstract description 4
- 239000001963 growth medium Substances 0.000 claims description 12
- 229960003957 dexamethasone Drugs 0.000 claims description 11
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- 101800003845 Neuropeptide Y Proteins 0.000 claims description 9
- 102400000064 Neuropeptide Y Human genes 0.000 claims description 9
- 108010019160 Pancreatin Proteins 0.000 claims description 9
- 229940055695 pancreatin Drugs 0.000 claims description 9
- 239000006285 cell suspension Substances 0.000 claims description 7
- ODBRNZZJSYPIDI-UHFFFAOYSA-N 3',4',5,7-tetrahydroxy-6-C-glucopyranosylflavone Natural products OC1C(O)C(O)C(CO)OC1C1=C(O)C=C(OC(=CC2=O)C=3C=C(O)C(O)=CC=3)C2=C1O ODBRNZZJSYPIDI-UHFFFAOYSA-N 0.000 claims description 6
- PLAPMLGJVGLZOV-UHFFFAOYSA-N Epi-orientin Natural products OC1C(O)C(O)C(CO)OC1C1=C(O)C=C(O)C2=C1OC(C=1C=C(O)C(O)=CC=1)=CC2=O PLAPMLGJVGLZOV-UHFFFAOYSA-N 0.000 claims description 6
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- ODBRNZZJSYPIDI-VJXVFPJBSA-N isoorientin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1C1=C(O)C=C(OC(=CC2=O)C=3C=C(O)C(O)=CC=3)C2=C1O ODBRNZZJSYPIDI-VJXVFPJBSA-N 0.000 claims description 6
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- PEFNSGRTCBGNAN-UHFFFAOYSA-N nephrocizin Natural products OC1C(O)C(O)C(CO)OC1OC1=CC(O)=C2C(=O)C=C(C=3C=C(O)C(O)=CC=3)OC2=C1 PEFNSGRTCBGNAN-UHFFFAOYSA-N 0.000 claims description 6
- 210000000130 stem cell Anatomy 0.000 claims description 6
- 239000001506 calcium phosphate Substances 0.000 claims description 5
- 229940078499 tricalcium phosphate Drugs 0.000 claims description 5
- 229910000391 tricalcium phosphate Inorganic materials 0.000 claims description 5
- 102100037241 Endoglin Human genes 0.000 claims description 4
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 claims description 4
- 101000881679 Homo sapiens Endoglin Proteins 0.000 claims description 4
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- 101000800116 Homo sapiens Thy-1 membrane glycoprotein Proteins 0.000 claims description 4
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- 102100024616 Platelet endothelial cell adhesion molecule Human genes 0.000 claims description 4
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- 239000002131 composite material Substances 0.000 claims description 4
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- 229910052588 hydroxylapatite Inorganic materials 0.000 claims description 4
- 229940055729 papain Drugs 0.000 claims description 4
- 235000019834 papain Nutrition 0.000 claims description 4
- 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 claims description 4
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 230000002607 hemopoietic effect Effects 0.000 claims description 2
- 150000004676 glycans Chemical class 0.000 claims 1
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 abstract description 4
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- 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
- 230000007547 defect Effects 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
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
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- 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
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 241000576429 Forsythia suspensa Species 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
- 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
- 229960005070 ascorbic acid Drugs 0.000 description 1
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- 239000011668 ascorbic acid Substances 0.000 description 1
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- 230000007423 decrease Effects 0.000 description 1
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- 210000004268 dentin Anatomy 0.000 description 1
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- 210000000981 epithelium Anatomy 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 239000003292 glue 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
- 229960000890 hydrocortisone Drugs 0.000 description 1
- 239000007943 implant Substances 0.000 description 1
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- 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
- 238000012360 testing method Methods 0.000 description 1
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Classifications
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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
- C12N5/0664—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
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- 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
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- 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
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- 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
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
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Abstract
The present invention belongs to the field of bio-medicine, and in particular relates to dental pulp stem cells, a preparation method thereof and a related bone tissue engineering material. The preparation method of dental pulp stem cells is as follows: taking pulp tissue, shearing the tissue, adding a digestion liquid, digesting at 37 DEG C, 200rpm for 10-20min, terminating the digestion by a serum-free medium, blowing and beating to discrete cell mass to obtain single discrete cells, centrifuging at 2000rpm for 5min, discarding a supernatant, washing twice in a serum-free medium, then adding serum-free medium to re-suspend the cells and adjusting the cell density to 1*10<3> / ml, culturing in a carbon dioxide incubator at 37 DEG C and with humidity of 95%, isolating digestive cells from the digestion liquid when the cells grow to 80%-90% confusion, and conducting subculture within a serum-free medium.
Description
Technical field
The invention belongs to field of pharmaceutical biology, be specifically related to a kind of dental pulp stem cell, preparation method and related bone tissue thereof
Engineering material.
Background technology
The defect of teeth caused due to a variety of causes and absence of tooth have become harm oral cavity or even health main
Disease, its incidence of disease increases year by year.Currently mainly it is by mean of various dental material filling to tackle tooth defect disease, although
The continuation that can stop pathology to a certain extent develops, but can not solve root problem.Although solving way for absence of tooth
Footpath is a lot, but most effective and the most basic way or the biological regeneration of tooth.
Along with the fast development of regeneration medical science, the effectively treatment for defect of teeth and absence of tooth brings hope.
The most existing many research and utilization Dental epitheliums and the mesochymal interaction of Odontogenic cysts have regenerated dental tissue.Commonly use
Odontogenic cysts mesenchyma includes dental papilla cells and Dental Pulp Cells.It is difficult to clinically due to dental papilla cells obtain, therefore,
The seed cell first-selection being 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 done
Cell.
Within 2000, being found that dental pulp stem cell first, Gronthos etc. finds to exist in vitro deciduous teeth and wisdom tooth
Dental pulp stem cell.This is that a class has high proliferation ability, highly self-renewal capacity, the cell of multinomial differentiation capability.Grind
Study carefully and utilize this cell successfully to build dental pulp, dentine and dental tissue.Dental pulp stem cell (dental pulp stem
Cell, DPSC) on biological characteristics, there is a lot of similarity with other tissue-derived stem cells.Gronthos etc. are to tooth
The cloning efficiency of marrow stem cell is studied, with bone marrow stroma stem cell (bone marrow stromal cell,
BMSC) find more afterwards: the cloning efficiency of the DPSC in tooth source is apparently higher than the BMSC of derived from bone marrow, and this indicates that
DPSC has compared with the higher Reproductive activity of BMSC and self-renewal capacity.And dental pulp stem cell is becoming fat, becoming cartilage and is becoming neural
Induction aspect all has research confirmation dental pulp stem cell to have the potential of Multidirectional Differentiation, implants dental pulp stem cell for it in vivo and makes to be subject to
Damage or the tissue of decline, organ improvement or recovery function provide theoretical foundation.
Summary of the invention
It is an object of the present invention to provide the preparation method of a kind of dental pulp stem cell, specifically comprise the following steps that
Taking pulp tissue, add tissue digestion liquid after shredding, 37 DEG C, 200rpm digests 10-20min, and serum free medium terminates
Digestion, piping and druming discrete cellular agglomerate obtains single discrete cell, and 2000rpm is centrifuged 5min, abandons supernatant, and serum free medium is clear
Wash twice, add serum free medium re-suspended cell and adjust cell density to 1 × 103Individual/ml, in 37 DEG C, humidity be
The CO2gas incubator of 95% is cultivated, when cell grows to 80%-90% fusion, after separating digesting liquid vitellophag
Use serum free medium Secondary Culture.
In one embodiment of the invention, described tissue digestion liquid is containing 1g/L pancreatin and 0.5g/L papain
D-Hanks liquid, pH value is 7.4.Described tissue digestion liquid is 10～16ml:1g with the envelope-bulk to weight ratio of pulp tissue.Described
Separating digesting liquid is the D-Hanks liquid containing 1g/L pancreatin, and pH value is 7.4.
In another embodiment of the present invention, described serum free medium is containing IGF 1-10ug/L, neuropeptide tyrosine
1-20ng/L, homo-orientin 1-20ug/L, the MEM culture medium of forsythin 1-20ug/L and dexamethasone 1-20mM.Further
In ground embodiment, described serum free medium is containing IGF 5ug/L, neuropeptide tyrosine 15ng/L, homo-orientin 5ug/L, the capsule of weeping forsythia
The MEM culture medium of glycosides 8ug/L and dexamethasone 5mM.
It is a further object to provide dental pulp stem cell prepared by above-mentioned preparation method, described dental pulp stem cell is several
Do not express label CD31 and CD34 specific to hemopoietic system；And to surface marker specific to mescenchymal stem cell
CD90 and CD105 has higher expression rate.
The three of the purpose of the present invention are to provide the preparation method of the Composite Bone timbering material loading above-mentioned dental pulp stem cell, tool
Body step is as follows:
1) dental pulp stem cell suspension is prepared；
2) bone holder material is immersed in dental pulp stem cell suspension, obtain Composite Bone timbering material.
In one embodiment of the invention, the preparation method of described bone holder material is: shitosan is dissolved in 2%
In acetum, stirring is completely dissolved to shitosan, filters to obtain the solution of weight 2% shitosan；Then in solution, add hydroxyl
Apatite and the water slurry of bata-tricalcium phosphate, be sufficiently stirred for 24h, is aged 1 day, filters to obtain precipitation, is existed by heat pressing forming machines
Suppressing 5min under conditions of 70 DEG C and 12MPa makes it be molded, and obtains bone holder material.Further, described shitosan, hydroxyl phosphorus
The weight ratio of lime stone and bata-tricalcium phosphate is 50:15:35.
In another embodiment of the present invention, the preparation method of described dental pulp stem cell suspension is: take pulp tissue,
Adding tissue digestion liquid after shredding, 37 DEG C, 200rpm digest 10-20min, and serum free medium terminates digesting, blow and beat discrete carefully
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 re-suspended cell also adjusts cell density to 1 × 103Individual/ml, in 37 DEG C, humidity be 95% carbon dioxide cultivate
Case is cultivated, when cell grows to 80%-90% fusion, passes on serum free medium after separating digesting liquid vitellophag
Cultivate, cultivate to collecting dental pulp stem cell during 3 generation, be configured to 1 × 10 with serum free medium5The dental pulp stem cell of individual/ml
Suspension.In further carrying out scheme, described serum free medium is containing IGF 5ug/L, neuropeptide tyrosine 15ng/L, different Polygonum orientale
Element 5ug/L, the MEM culture medium of forsythin 8ug/L and dexamethasone 5mM.Described tissue digestion liquid is containing 1g/L pancreatin and 0.5g/
The D-Hanks liquid of L papain, pH value is 7.4.The envelope-bulk to weight ratio of described tissue digestion liquid and pulp tissue be 10～
16ml:1g.Described separating digesting liquid is the D-Hanks liquid containing 1g/L pancreatin, and pH value is 7.4.
Detailed description of the invention
To further describe the present invention below in detail.It is pointed out that following description is only to application claims
The illustration of the technical scheme of protection, the not any restriction to these technical schemes.Protection scope of the present invention is with appended
The content that claims are recorded is as the criterion.
Embodiment 1 dental pulp stem cell is cultivated
Pulp tissue, the pulp tissue of excision root tip 1mm is gripped with aseptic nipper；With ophthalmology curved scissors, pulp tissue is cut into
1mm3, it is placed in 50mL centrifuge tube, adds the tissue digestion liquid of 13ml (containing 1g/L pancreatin and the D-of 0.5g/L papain
Hanks liquid, pH value is 7.4), it is sufficiently mixed after sealing uniformly, is transferred in Tempeerature-constant air shaking table, 37 DEG C, 200rpm digestion
15min ；Add isopyknic serum free medium and repeatedly blow and beat discrete cellular agglomerate, by the cell screen filtration of 70 μm,
2000rpm is centrifuged 3min；Utilizing PBS to clean 1～2 time, precipitation uses serum free medium (containing IGF 5ug/L, neuropeptide tyrosine
15ng/L, homo-orientin 5ug/L, the MEM culture medium of forsythin 8ug/L and dexamethasone 5mM, lower same) resuspended, with 1 × 103
In individual/ml inoculated and cultured ware, mix cell suspension, be put in 37 DEG C, humidity be 95% CO2gas incubator in cultivate；24h
After change liquid and remove non-attached cell, within the most every 3 days, change liquid 1 time, when cell grows to 80%～90% converges, separating digesting liquid
(the D-Hanks liquid containing 1g/L pancreatin, pH value is 7.4) vitellophag, carries out Secondary Culture.
Embodiment 2
According to the cultural method of embodiment 1, P1 for time, use Trypan Blue to carry out cell viability and concentration measure, specifically
For with the dental pulp stem cell in 0.1% trypsinization liquid collection culture dish, (in each culture dish, original paving is done into 10000 dental pulps
Cell), PBS, then dyes with the trypan blue solution of 0.4%, calculates living cells number (parallel examination in each culture dish
Test three times), and by flow cytomery label CD31, CD34, CD90 and CD105.
Result is as follows:
The cultural method of comparative example 1 is with embodiment 1, and the tissue digestion liquid differing only in comparative example 1 is containing 1.5g/L pancreatin
D-Hanks liquid, pH value is 7.4；
The cultural method of comparative example 2, with embodiment 1, differs only in the tissue digestion liquid of comparative example 2 for containing 1.5g/L I type glue
The D-Hanks liquid of protoenzyme, pH value is 7.4；
The cultural method of comparative example 3 is with embodiment 1, and the serum free medium differing only in comparative example 3 is containing IGF 5ug/L,
The MEM culture medium of neuropeptide tyrosine 15ng/L and dexamethasone 5mM
The cultural method of comparative example 4 is with embodiment 1, and the serum free medium differing only in comparative example 4 is containing IGF 5ug/L,
Neuropeptide tyrosine 15ng/L, the MEM culture medium of homo-orientin 13ug/L and dexamethasone 5mM
The cultural method of comparative example 5 is with embodiment 1, and the serum free medium differing only in comparative example 5 is containing IGF 5ug/L,
Neuropeptide tyrosine 15ng/L, the MEM culture medium of forsythin 13ug/L and dexamethasone 5mM
The cultural method of comparative example 6 is with embodiment 1, and the serum free medium differing only in comparative example 5 is Chinese patent
Serum free medium disclosed in CN201510095430 specification embodiment 1: SITE100* (sigma) 10ml, ascorbic acid
More than 300mM, PDGF 5ug, hydrocortisone 5ug, EGF3ng, b-FGF, 3ng, PTH 100ng, dexamethasone 10mM, IMDM
Amount
Through flow cytomery, each group stem cell all shows the marker feature of dental pulp stem cell, the table of CD31 and CD34
The rate that reaches is less than 0.5%；The expression rate of CD90 and CD105 is above 98%.
Embodiment 3 Osteoblast Differentiation ability is identified
Take the dental pulp stem cell that P3 obtains close to the embodiment 1 converged for well-grown respectively, use serum-free with after trypsinization
Culture medium (embodiment 1) counts after making cell suspension.Adjusting cell density is 1 × 105/ mL, 2mL/ insert in hole in 6 orifice plates.
When cell length to 80% merges, discarding nutrient solution, (dimension is raw for DMEM nutrient solution, sodium glycero-phosphate to be changed to Osteogenic Induction Medium
Element C and dexamethasone).Continuous induction, replacing in every 3 days once induces liquid, stops induction at the 28th day, collects cell, and cracking carries
Take protein determination alkaline phosphatase, draw culture medium and measure BGP (according to the method mensuration of commercial reagent box, often group mensuration 5
Individual sample).
Result is as follows:
Embodiment 4 loads the bone holder material of dental pulp stem cell and prepares
Being dissolved in the acetum of 2% by 50g shitosan, stirring is completely dissolved to shitosan, filters to obtain weight 2% shitosan molten
Liquid；Then it is added drop-wise in solution in the water slurry containing 15g hydroxyapatite and 35g bata-tricalcium phosphate, is sufficiently stirred for 24h,
It is aged 1 day, filters to obtain precipitation, under conditions of 70 DEG C and 12MPa, suppress 5min by heat pressing forming machines and make it be molded, obtain bone
Timbering 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), stand 24h, be then transferred to bone holder material in 6 hole culture dishes train with serum free medium (embodiment 1)
Supporting, changed liquid once every 3 days, after cultivating 2 weeks, the cell on trypsinization bone holder material, Trypan Blue detection cell is lived
Property, result shows, dental pulp stem cell well-grown (cytoactive rate is 99%) on bone holder material, and beat all
It is that, under conditions of not having osteogenic induction agent, the dental pulp stem cell on bone support shows obvious Osteoblast Differentiation characteristic (alizarin
Red colouring finds obvious calcium tubercle, and content of alkaline phosphatase is 101 ± 13U/g；In culture medium BGP content be 0.15 ±
0.03).
Present invention merely illustrates some claimed specific embodiments, one of them or more skill
Technical characteristic described in art scheme can be combined with arbitrary one or more technical schemes, and these are combined and obtain
Technical scheme also in the application protection domain, combined just as these and the technical scheme that obtains is open in the present invention
In content as concrete record.
Claims (8)
1. a preparation method for dental pulp stem cell, specifically comprises the following steps that
Taking pulp tissue, add tissue digestion liquid after shredding, 37 DEG C, 200rpm digests 10-20min, and serum free medium terminates
Digestion, piping and druming discrete cellular agglomerate obtains single discrete cell, and 2000rpm is centrifuged 5min, abandons supernatant, and serum free medium is clear
Wash twice, add serum free medium re-suspended cell and adjust cell density to 1 × 103Individual/ml, in 37 DEG C, humidity be
The CO2gas incubator of 95% is cultivated, when cell grows to 80%-90% fusion, after separating digesting liquid vitellophag
Use serum free medium Secondary Culture.
The preparation method of dental pulp stem cell the most according to claim 1, it is characterised in that described tissue digestion liquid is for containing
The D-Hanks liquid of 1g/L pancreatin and 0.5g/L papain, pH value is 7.4, described tissue digestion liquid and the body of pulp tissue
Long-pending weight ratio is 10～16ml:1g.
The preparation method of dental pulp stem cell the most according to claim 1, it is characterised in that described separating digesting liquid is for containing
The D-Hanks liquid of 1g/L pancreatin, pH value is 7.4.
The preparation method of dental pulp stem cell the most according to claim 1, it is characterised in that described serum free medium is for containing
IGF 1-10ug/L, neuropeptide tyrosine 1-20ng/L, homo-orientin 1-20ug/L, forsythin 1-20ug/L and dexamethasone 1-20mM
MEM culture medium.
The preparation method of dental pulp stem cell the most according to claim 4, it is characterised in that described serum free medium is for containing
IGF 5ug/L, neuropeptide tyrosine 15ng/L, homo-orientin 5ug/L, the MEM culture medium of forsythin 8ug/L and dexamethasone 5mM.
6. the dental pulp stem cell that prepared by the preparation method of the dental pulp stem cell described in claim 1-5, described dental pulp is dry thin
Born of the same parents express label CD31 and CD34 specific to hemopoietic system hardly；And to surface markers specific to mescenchymal stem cell
Thing CD90 and CD105 has higher expression rate.
7. load a preparation method for the Composite Bone timbering material of dental pulp stem cell described in any one of claim 1-5, specifically
Step is as follows:
1) dental pulp stem cell suspension is prepared；
2) bone holder material is immersed in dental pulp stem cell suspension, obtain Composite Bone timbering material.
Preparation method the most according to claim 7, it is characterised in that the preparation method of described bone holder material is: by shell
Glycan is dissolved in the acetum of 2%, and stirring is completely dissolved to shitosan, filters to obtain the solution of weight 2% shitosan；Then to molten
Liquid adds hydroxyapatite and the water slurry of bata-tricalcium phosphate, is sufficiently stirred for 24h, is aged 1 day, filter to obtain precipitation, pass through
Heat pressing forming machines suppresses 5min under conditions of 70 DEG C and 12MPa makes it be molded, and obtains bone holder material, further, described
The weight ratio of shitosan, hydroxyapatite and bata-tricalcium phosphate is 50:15:35.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN110438069A (en) * | 2019-08-22 | 2019-11-12 | 石学高 | A kind of phillygenol for promoting human adipose mesenchymal stem cells at the purposes of cartilage differentiation in vitro |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101717750A (en) * | 2009-12-09 | 2010-06-02 | 中国人民解放军第四军医大学 | Method for constructing banks of human dental pulp stem cells |
| US20120148536A1 (en) * | 2010-12-14 | 2012-06-14 | Morehouse School Of Medicine | Methods and storage and retrieval of functional mature retinal cells |
| CN102860884A (en) * | 2012-09-17 | 2013-01-09 | 清华大学 | Tissue engineered osteochondral composite stent and preparation method thereof |
| CN104711219A (en) * | 2015-03-01 | 2015-06-17 | 安徽新生命干细胞科技有限公司 | Dental pulp stem cell culture medium |
| CN105087474A (en) * | 2015-04-27 | 2015-11-25 | 广州赛莱拉干细胞科技股份有限公司 | Culture method of deciduous tooth pulp stem cells |
| CN105087462A (en) * | 2015-07-24 | 2015-11-25 | 广州赛莱拉干细胞科技股份有限公司 | Stem cell freezing medium and stem cell freezing method |
-
2016
- 2016-03-08 CN CN201610127648.4A patent/CN105754934B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101717750A (en) * | 2009-12-09 | 2010-06-02 | 中国人民解放军第四军医大学 | Method for constructing banks of human dental pulp stem cells |
| US20120148536A1 (en) * | 2010-12-14 | 2012-06-14 | Morehouse School Of Medicine | Methods and storage and retrieval of functional mature retinal cells |
| CN102860884A (en) * | 2012-09-17 | 2013-01-09 | 清华大学 | Tissue engineered osteochondral composite stent and preparation method thereof |
| CN104711219A (en) * | 2015-03-01 | 2015-06-17 | 安徽新生命干细胞科技有限公司 | Dental pulp stem cell culture medium |
| CN105087474A (en) * | 2015-04-27 | 2015-11-25 | 广州赛莱拉干细胞科技股份有限公司 | Culture method of deciduous tooth pulp stem cells |
| CN105087462A (en) * | 2015-07-24 | 2015-11-25 | 广州赛莱拉干细胞科技股份有限公司 | Stem cell freezing medium and stem cell freezing method |
Non-Patent Citations (4)
| Title |
|---|
| 李珊等: "成人牙髓干细胞与壳聚糖-磷酸三钙复合材料相容性试验研究", 《口腔医学研究》 * |
| 杜晓岩等: "羟基磷灰石-磷酸三钙/壳聚糖骨碎补复合材料修复骨缺损的实验研究", 《中国美容医学》 * |
| 贺慧霞等: "两种钙磷材料表面壳聚糖修饰对人牙髓干细胞黏附和增殖的影响", 《牙体牙髓牙周病学杂志》 * |
| 鞠躬等: "《神经生物学》", 30 September 2015, 第四军医大学出版社 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN110438069A (en) * | 2019-08-22 | 2019-11-12 | 石学高 | A kind of phillygenol for promoting human adipose mesenchymal stem cells at the purposes of cartilage differentiation in vitro |
| 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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