CN108373993A - The method that inducing bone mesenchymal stem cell directional is divided into osteoblast - Google Patents
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Abstract
The invention belongs to biomedical engineering fields, and in particular to a kind of method that inducing bone mesenchymal stem cell directional is divided into osteoblast.Lack a kind of method of easy, economic, safe and effective inducing bone mesenchymal stem cell to osteoblast differentiation in the prior art, the present invention provides a kind of method that inducing bone mesenchymal stem cell directional is divided into osteoblast.The present invention builds polymer three-dimensional structure, provides suitable microenvironment, then use collagen composite, the Cascaded amplification of signal between mediated cell；It is placed in Osteogenic Induction Medium and cultivates again, strengthen the expression of skeletonization associated transcription factor, promote bone mesenchymal stem cell to osteoblast differentiation.
Description
Technical field
The invention belongs to biomedical engineering fields, and in particular to a kind of inducing bone mesenchymal stem cell directional is divided into
The method of osteoblast.
Background technology
Mesenchymal stem cell (Bone mesenchymal stem cells, BMSCs) is a kind of adult stem cell,
Can self-renewing, have multi-lineage potential, under inductive condition appropriate can with directed differentiation become cartilage, skeletonization, flesh
The Various Tissues cell such as meat, fat, nerve.MSCs becomes preclinical medicine and the reparation of clinical medicine injuries of tissues and organs and regeneration neck
The hot spot of domain research, but there is also some technical problems at present, as differentiation efficiency is low or spontaneous differentiation forms foreign cell
Even abnormal differentiation is at tumour cell.In bone tissue engineer application, it is to utilize it that directional induction MSCs, which is divided into osteoblast,
Build a committed step of bone tissue engineer.
Currently used abductive approach mainly has：One, exogenous inducible factor differentiation of stem cells is introduced.This method
The problem of it is complicated to operate, induced efficiency is low, expensive, discharges difficult control, is unfavorable for larger scale clinical popularization and application.Two, will
Certain albumen or the recombinant DNA of growth factor import stem cell by transgenic technology, induce its directed differentiation.Using transgenosis
Although technology improves differentiation efficiency, reduce cost, but due to the problems such as there are safety, moral checks, also away from clinical application
There is prodigious distance.Therefore, a kind of easy, economic, safe and effective abductive approach is established, is induction stem cell to skeletonization
Direction breaks up and the key point of tissue-engineered bone structure.
The microenvironment of MSCs provides crucial biochemistry and physical signal to start or maintaining stem cell to break up.Study table
It is bright：The complicated chemical microenvironment that the chemical signal Signal Transduction Pathways of the compositions such as hormone, growth factor, cell factor are formed can regulate and control
The differentiation destiny of mesenchymal stem cell；Equally, suitable physics microenvironment, mechanical property, surface shape such as basis material
Looks etc. also play very important regulating and controlling effect in stem cell atomization.
Culture medium has been acknowledged as external evoked at present dry as the important regulatory factor of extracellular chemical micro-environment
Cell directional breaks up one of simple, effective method, common induction medium includes skeletonization, at cartilage, at fat and at nerve-inducing
Medium etc..There is numerous studies report in various tissue repairs currently with induction medium regulation and control stem cell directional differentiation.
Human marrow mesenchymal stem cell is inoculated in gelfoam by Ponticiello etc., is cultivated 3 weeks in chondrocyte induction medium, raw
At cartilaginous tissue [Ponticiello MS, Schinaql RM, Kadiyala S, Barry FP.Gelatin-based
resorbable sponge as a carrier matrix for human mesenchymal stem cells in
cartilage regeneration therapy.J Biomed Mater Res 2000；52:246~255.].At cardiac muscle
Under the action of induction medium, mescenchymal stem cell can myocardiac differentiation, this is for the clinical cardiac muscle for substituting necrosis or apoptosis
Cell promotes heart function recovery to have very tempting potential applicability in clinical practice.But since induction medium is mainly used in vitro culture,
There is larger limitation, so limit extensive use of the induction medium in organizational project.In addition, induction medium is to stem cell
The regulatory mechanism of differentiation process has larger difference compared with human body natural's process.
Other than the exogenous chemical signal in induction medium, essential host material is as dry thin in organizational project
The important component of born of the same parents' microenvironment plays important regulating and controlling effect simultaneously to the differentiation destiny of stem cell.When stem cell and base
When material interacts, host material directly affects sticking and being aggregated for stem cell, and passes through friendship between the stem cell being aggregated
The synergistic effect stimulation peripheral cell of mutual response secretes the specific cells factor or molecular signal, and signal cascade is caused to amplify, to
Influence the differentiation pathway and destiny of stem cell.The bio-active group of material can equally influence the differentiation of stem cell, especially when
When being used as host material using extracellular matrix (Extracellular Matrix, ECM), the basic act to stem cell is even more
Play conclusive influence.The Richard O.Hynes professors of Massachusetts science and engineering detail carefully in a survey article of Science
Extracellular matrix influences the potential mechanism of stem cell behavior and differentiation process by biochemical signals：Extracellular matrix passes through anchoring
Integral protein mediates biochemical signals transmission (inside-out signaling) from inside to outside and ecto-entad biochemical signals to transmit
(outside-in signaling), to influence the expression of related gene, regulating cell breaks up [Hynes
RO.Theextracellular matrix:Not just pretty fibrils.Science 2009；326:1216~
1219].Other than biochemical signals, the mechanical signal that the mechanics microenvironment and cell mechanical force of intraor extracellular generate also may be used
Directly affect the self-renewing and differentiation of stem cell.Mechanical stimulation can activate stem cell surface receptor and focal adhension, and then touch
Hair intracellular signal cascades amplification is converted into biological signals, causes a series of biologically, final influence stem cell
Break up destiny, MSC is particularly sensitive to the elasticity of material surface.The classical article of one of Cell in 2006 just once reports based elastic
Modulus plays a decisive role to stem cell differentiation destiny：There are no the conditions of chemical inducer for Engler et al. researchs hair
Under, by stem cell culture in the matrix of 25~40kPa (simulation osteoid hardness), stem cell presents similar with osteoblast
Polygonal, and the up-regulation of osteoblast marker molecule Runx2 expressions [Engler AJ, SenS, Sweeney HL,
DischerDE.Matrix elasticity directs stem cell lineage specification.Cell
2006；126:677~689.].
The differentiation of MSCs and the microenvironment of growth are closely related, but there is presently no it is a kind of it is easy, economical, it is safe and
It is osteoblast that precisely effective abductive approach, which can direct it induction,.
Invention content
The technical problem to be solved in the present invention is：Lack a kind of easy, economic, safe and effective induction in the prior art
The method of bone mesenchymal stem cell to osteoblast differentiation.
The present invention provides a kind of method that inducing bone mesenchymal stem cell directional is divided into osteoblast, including following step
Suddenly：
A, degradable medical high molecular material and the active matter of the adherency of promotion osteoblast and proliferation is compound, preparation has
The film of three-dimensional structure；
B, surface-functionalized processing is carried out to the film of three-dimensional structure；
C, using the active matter with osteocyte adherency and proliferation activity to the thin of the three-dimensional structure of surface-functionalized processing
Film carries out surface modification；
D, it is obtained after the film of the modified three-dimensional structure in surface in c being washed, dried for being filled between inducing bone marrow
Matter stem cell directional is divided into the three-dimensional structure of osteoblast.
E, after mesenchymal stem cell being inoculated in the three-dimensional structure described in step d preculture 4~48 hours, add
Enter Osteogenic Induction Medium culture, obtains osteoblast.
Wherein, above-mentioned inducing bone mesenchymal stem cell directional is divided into the method for osteoblast, can described in step a
Degradation medical macromolecular materials include the aliphatic polyester of medical grade, polylactic acid, polycaprolactone, poly lactic-co-glycolic acid copolymerization
One or several kinds of combinations of object, chitosan, cellulose, polyvinyl alcohol, the work for promoting osteoblast adherency and proliferation
Property object include collagen, gelatin, polypeptide, albumen, growth factor, chitosan, nanometer hydroxyapatite and tricalcium phosphate it is a kind of or
Several combinations.
Wherein, above-mentioned inducing bone mesenchymal stem cell directional is divided into the method for osteoblast, in the step a,
It is using method of electrostatic spinning that degradable medical high molecular material and the active matter of the adherency of promotion osteoblast and proliferation is compound, it prepares
Film with three-dimensional structure.
Wherein, above-mentioned inducing bone mesenchymal stem cell directional is divided into the method for osteoblast, in the step b
" carrying out surface-functionalized processing to the film of three-dimensional structure " is specially：By the film of three-dimensional structure be put into pH value be 7.5~
In the dopamine Tris-HCl solution of 9.0, a concentration of 0.1wt%~2wt%, 1-48h is reacted.
Wherein, above-mentioned inducing bone mesenchymal stem cell directional is divided into the method for osteoblast, in the step c
" surface is carried out to the film of the three-dimensional structure of surface-functionalized processing using the active matter with osteocyte adherency and proliferation activity
It is modified " reaction condition be：The concentration of active matter with osteocyte adherency and proliferation activity is not higher than 10wt%, the temperature of reaction
Degree is 0~60 DEG C, and the reaction time is less than 48h.
Wherein, above-mentioned inducing bone mesenchymal stem cell directional is divided into the method for osteoblast, and the step b is also wrapped
It includes：Before carrying out surface-functionalized processing to the film of three-dimensional structure, to the film of the cleaning three-dimensional structure prepared in step a into
Row cleaning removes solvent, is dried.
Wherein, above-mentioned inducing bone mesenchymal stem cell directional is divided into the method for osteoblast, the three-dimensional structure
Film in, it is described promote osteoblast adherency and proliferation active matter content be 0.05wt%~20wt%.
Preferably, the active matter for promoting osteoblast to adhere to and be proliferated is collagen, and the collagen is type i collagen,
Collagen concentration is 1~15mg/ml, and the pH value of collagen is 4~6.5.
It is highly preferred that the collagen concentration described in step b is 7~9mg/ml, pH value is 5~6.5.
Wherein, above-mentioned inducing bone mesenchymal stem cell directional is divided into the method for osteoblast, described in step e
Pre-incubation time is 4~24 hours.
Wherein, above-mentioned inducing bone mesenchymal stem cell directional is divided into the method for osteoblast, described in step e
Osteogenic Induction Medium forms：Dexamethasone 10-5~10-2mmol/L, 5~20mmol/L of sodium β-glycerophosphate resist
0.02~0.05mmol/L of bad hematic acid.
Beneficial effects of the present invention are：The present invention provides a kind of inducing bone mesenchymal stem cell directionals to be divided into skeletonization
The method of cell, by the thin-film material for preparing three-dimensional structure and strong osteogenic activity.The adherency of osteoblast is to determine that it is follow-up
The first step of increment and bone formation performance, so the high expression of the collagen on surface, growth factor etc., enables to osteoblast in material
Material surface short time interior suction echos growth.By mixed and modified method material there is table preparation to contain the thin of osteogenic activity object
Film, with the degradation of polymer, active matter gradually discharges, and realizes good bone formation performance；This method is significantly reduced work simultaneously
Influence of the property material mixing to the mechanics of materials.The method of the present invention is easy to operate, economical, safe, precisely between effective regulation and control marrow
Mesenchymal stem cells have important clinical value to Osteoblast Differentiation in bone disorder treatment, bone graft technique.
Description of the drawings
Fig. 1 Elisa methods are under the conditions of osteogenic induction, the differentiation early stage alkali in polymer three-dimensional body structure surface growth course
Acid phosphatase (ALP), and break up the testing result of late period osteocalcin (OCN), unit ng/ml；Indicated respectively in figure 2 days, 4 days, 6
It, the block diagrams of 14 days results be followed successively by polymer three-dimensional structure+ordinary culture medium from left to right, polymer three-dimensional structure+at
Self-bone grafting culture medium.
Fig. 2 Elisa methods are under the conditions of osteogenic induction, skeletonization of the BMSC in polymer three-dimensional body structure surface growth course
Break up the testing result of the early transcription factor, unit ng/ml.A.Dlx5；B.Osterix.Indicated respectively in figure 2 days, 4 days, 6
It, the block diagrams of 14 days results be followed successively by polymer three-dimensional structure+ordinary culture medium from left to right, polymer three-dimensional structure+at
Self-bone grafting culture medium.
Specific implementation mode
The present invention provides a kind of method that inducing bone mesenchymal stem cell directional is divided into osteoblast, including following step
Suddenly：
A, degradable medical high molecular material and the active matter of the adherency of promotion osteoblast and proliferation is compound, preparation has
The film of three-dimensional structure；
B, surface-functionalized processing is carried out to the film of three-dimensional structure；
C, using the active matter with osteocyte adherency and proliferation activity to the thin of the three-dimensional structure of surface-functionalized processing
Film carries out surface modification；
D, it is obtained after the film of the modified three-dimensional structure in surface in c being washed, dried for being filled between inducing bone marrow
Matter stem cell directional is divided into the three-dimensional structure of osteoblast.
E, after mesenchymal stem cell being inoculated in the three-dimensional structure described in step d preculture 4~48 hours, add
Enter Osteogenic Induction Medium culture, obtains osteoblast.
Wherein, above-mentioned inducing bone mesenchymal stem cell directional is divided into the method for osteoblast, can described in step a
Degradation medical macromolecular materials include the aliphatic polyester of medical grade, polylactic acid, polycaprolactone, poly lactic-co-glycolic acid copolymerization
One or several kinds of combinations of object, chitosan, cellulose, polyvinyl alcohol, the work for promoting osteoblast adherency and proliferation
Property object include collagen, gelatin, polypeptide, albumen, growth factor, chitosan, nanometer hydroxyapatite and tricalcium phosphate it is a kind of or
Several combinations.
Wherein, above-mentioned inducing bone mesenchymal stem cell directional is divided into the method for osteoblast, in the step a,
It is using method of electrostatic spinning that degradable medical high molecular material and the active matter of the adherency of promotion osteoblast and proliferation is compound, it prepares
Film with three-dimensional structure.
Wherein, above-mentioned inducing bone mesenchymal stem cell directional is divided into the method for osteoblast, in the step b
" carrying out surface-functionalized processing to the film of three-dimensional structure " is specially：By the film of three-dimensional structure be put into pH value be 7.5~
In the dopamine Tris-HCl solution of 9.0, a concentration of 0.1wt%~2wt%, 1-48h is reacted.
Wherein, above-mentioned inducing bone mesenchymal stem cell directional is divided into the method for osteoblast, in the step c
" surface is carried out to the film of the three-dimensional structure of surface-functionalized processing using the active matter with osteocyte adherency and proliferation activity
It is modified " reaction condition be：The concentration of active matter with osteocyte adherency and proliferation activity is not higher than 10wt%, the temperature of reaction
Degree is 0~60 DEG C, and the reaction time is less than 48h.
Wherein, above-mentioned inducing bone mesenchymal stem cell directional is divided into the method for osteoblast, and the step b is also wrapped
It includes：Before carrying out surface-functionalized processing to the film of three-dimensional structure, to the film of the cleaning three-dimensional structure prepared in step a into
Row cleaning removes solvent, is dried.
Wherein, above-mentioned inducing bone mesenchymal stem cell directional is divided into the method for osteoblast, the three-dimensional structure
Film in, it is described promote osteoblast adherency and proliferation active matter content be 0.05wt%~20wt%.
Preferably, the active matter for promoting osteoblast to adhere to and be proliferated is collagen, and the collagen is type i collagen,
Collagen concentration is 1~15mg/ml, and the pH value of collagen is 4~6.5.
It is highly preferred that the collagen concentration described in step b is 7~9mg/ml, pH value is 5~6.5.
Wherein, above-mentioned inducing bone mesenchymal stem cell directional is divided into the method for osteoblast, described in step e
Pre-incubation time is 4~24 hours.
Wherein, above-mentioned inducing bone mesenchymal stem cell directional is divided into the method for osteoblast, described in step e
Osteogenic Induction Medium forms：Dexamethasone 10-5~10-2mmol/L, 5~20mmol/L of sodium β-glycerophosphate resist
0.02~0.05mmol/L of bad hematic acid.
Embodiment
Explanation is further explained to the specific implementation mode of the present invention below by embodiment.Technology in the art
Personnel are, it will be appreciated that the technology disclosed in following examples represents the technology that can be used for implementing the present invention of inventor's discovery, therefore
The preferred embodiment for implementing the present invention can be considered as.But those skilled in the art should be understood that here according to this specification
Disclosed specific embodiment can make many modifications, still can obtain identical or similar as a result, rather than away from this hair
Bright spirit or scope.
Unless otherwise defined, the term of all technologies as used herein and science, and the technology in fields of the present invention
Personnel institute is normally understood equivalent in meaning, and being disclosed reference and their materials of reference will all be incorporated.
Those skilled in the art will recognize or just will appreciate that by routine test many described here
Invention particular embodiment many equivalent technologies.These will equally be comprised in claims.
In embodiment, the ordinary culture medium group becomes：α-MEM trainings containing 89ml in per 100ml ordinary culture mediums
Support base, the fetal calf serum of 10ml and 1ml's is dual anti-.
The Osteogenic Induction Medium group becomes：87.4ml α-the MEM contained in Osteogenic Induction Medium per 100ml
Culture medium, the fetal calf serum of 10ml and dual anti-, the 500ul vitamin Cs (50umol/L) of 1ml, the sodium glycero-phosphate of 1ml
(100mmol/L), 10ul dexamethasone (100nmol/L).
1 polymer three-dimensional structure of embodiment prepares the method with induced osteogenesis
1gPLGA, 10% chitosan are weighed, is put into trifluoroethanol solution, magnetic agitation is uniformly dissolved.It is just in parameter
18.5kv, negative pressure 2.8kv, humidity 35% is pressed to prepare the film with three-dimensional structure by electrospinning process at 19 DEG C of temperature,
Gained film is placed in vacuum drying chamber dry 48h, removes solvent；The thin-film material with three-dimensional structure of acquisition is carried out
The surface-functionalized processing of dopamine is 8.5 in pH value, in a concentration of 0.3wt% dopamines Tris-HCl solution, after reaction for 24 hours
It is washed with deionized 3 times.It is washed with deionized 3 times after pre-processing 1h in 2% glutaraldehyde.It is molten in the collagen of 5wt%
In liquid, reaction for 24 hours, controlled at 37 DEG C, is finally washed with deionized 3 times, is put into vacuum drying chamber dry 48h and obtains
Polymer three-dimensional structure.After mesenchymal stem cell is seeded in polymer three-dimensional body structure surface, ordinary culture medium, training is added
After supporting 14 days, the case where it is to Osteoblast Differentiation is investigated.
2 polymer three-dimensional structure of embodiment prepares the method with induced osteogenesis
1gPLGA, 10% chitosan are weighed, is put into trifluoroethanol solution, magnetic agitation is uniformly dissolved.It is just in parameter
18.5kv, negative pressure 2.8kv, humidity 35% is pressed to prepare the film with three-dimensional structure by electrospinning process at 19 DEG C of temperature,
Gained film is placed in vacuum drying chamber dry 48h, removes solvent；The thin-film material with three-dimensional structure of acquisition is carried out
The surface-functionalized processing of dopamine is 8.5 in pH value, in a concentration of 0.3wt% dopamines Tris-HCl solution, after reaction for 24 hours
It is washed with deionized 3 times.It is washed with deionized 3 times after pre-processing 1h in 2% glutaraldehyde.It is molten in the collagen of 5wt%
In liquid, reaction for 24 hours, controlled at 37 DEG C, is finally washed with deionized 3 times, is put into vacuum drying chamber dry 48h and obtains
Polymer three-dimensional structure.After mesenchymal stem cell is seeded in polymer three-dimensional body structure surface, osteogenic induction culture is added
Base investigates the case where it is to Osteoblast Differentiation after cultivating 14 days.
Compliance test result test
Cell differentiation
Polymer three-dimensional structure prepared by embodiment 1 and embodiment 2 is placed in 24 well culture plates.Take third generation growth prosperous
2*10 is made in the mesenchymal stem cell of the SD rats of Sheng4Cell suspension, be inoculated in three-dimensional structure table by the amount in the holes 1ml/
Face, embodiment 1 are cultivated in ordinary culture medium, and embodiment 2 is cultivated in Osteogenic Induction Medium.Culture 2 days respectively, 4 days, 6
It and taken out after 14 days, add 10ul lysates (Triton X-100) per hole, blow and beat repeatedly, and be positioned over mistake in 37 DEG C of incubators
At night, whether there is or not intact cells under the microscope, illustrate to operate by ELisa kits (Lan Ji, China), measure its extinction at 450 nm
Degree calculates Dlx5, Osterix, ALP and OCN value according to standard curve.The result is shown in Figure 1,2.
What Fig. 1 was investigated is alkaline phosphatase and osteocalcin of the mesenchymal stem cell in polymer three-dimensional body structure surface
Expression.As seen from Figure 1：Osteogenic induction medium is added in embodiment 2, alkaline phosphatase and the table of osteocalcin can be promoted
It reaches, shows that the addition of osteogenic induction medium can act synergistically with polymer three-dimensional structure, further strengthen polymer three-dimensional knot
The osteogenic induction ability of structure.
What Fig. 2 was investigated is influence of the different cultural methods to early stage skeletonization associated transcription factor Dlx5 and Osterix.By scheming
2 it is found that the osteogenic induction medium that embodiment 2 is added can remarkably promote the expression of Dlx5 and Osterix, further prove calcium phosphate
There is collaboration facilitation to stem cell Osteoblast Differentiation with osteogenic induction medium.
All references mentioned in the present invention is incorporated herein by reference, independent just as each document
It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can
To be made various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims
It encloses.
Claims (11)
1. the method that inducing bone mesenchymal stem cell directional is divided into osteoblast, which is characterized in that include the following steps：
A, degradable medical high molecular material and the active matter of the adherency of promotion osteoblast and proliferation is compound, it prepares with three-dimensional
The film of structure；
B, surface-functionalized processing is carried out to the film of three-dimensional structure；
C, using the active matter with osteocyte adherency and proliferation activity to the film of the three-dimensional structure of surface-functionalized processing into
Row surface is modified；
D, it obtains doing for inducing bone mesenchymal after the film of the modified three-dimensional structure in surface in c being washed, dried
Cell directional is divided into the three-dimensional structure of osteoblast.
E, after mesenchymal stem cell being inoculated in the three-dimensional structure described in step d preculture 4~48 hours, be added at
Self-bone grafting medium culture, obtains osteoblast.
2. the method that inducing bone mesenchymal stem cell directional according to claim 1 is divided into osteoblast, feature
It is：Degradable medical high molecular material described in step a includes the aliphatic polyester of medical grade, and polylactic acid, is gathered polycaprolactone
One or several kinds of combinations of poly lactic coglycolic acid, chitosan, cellulose, polyvinyl alcohol, the promotion skeletonization are thin
Born of the same parents adhere to and the active matter of proliferation includes collagen, gelatin, polypeptide, albumen, growth factor, chitosan, nanometer hydroxyapatite and
The combination of tricalcium phosphate one or several kinds.
3. the method that inducing bone mesenchymal stem cell directional according to claim 1 is divided into osteoblast, feature
It is：In the step a, degradable medical high molecular material is adhered to and is proliferated with promotion osteoblast using method of electrostatic spinning
Active matter it is compound, prepare with three-dimensional structure film.
4. the method that inducing bone mesenchymal stem cell directional according to claim 1 is divided into osteoblast, feature
It is：" carrying out surface-functionalized processing to the film of three-dimensional structure " in the step b is specially：The film of three-dimensional structure is put
It is 7.5~9.0 to enter pH value, in the dopamine Tris-HCl solution of a concentration of 0.1wt%~2wt%, reacts 1-48h.
5. the method that inducing bone mesenchymal stem cell directional according to claim 1 is divided into osteoblast, feature
It is：" three-dimensional of surface-functionalized processing is tied using the active matter with osteocyte adherency and proliferation activity in the step c
The film of structure carries out surface modification " reaction condition be：The concentration of active matter with osteocyte adherency and proliferation activity is not high
It it is 0~60 DEG C in the temperature of 10wt%, reaction, the reaction time is less than 48h.
6. the method that inducing bone mesenchymal stem cell directional according to claim 1 is divided into osteoblast, feature
It is：The step b further includes：It is clear to being prepared in step a before carrying out surface-functionalized processing to the film of three-dimensional structure
The film for washing three-dimensional structure is cleaned, except solvent, drying process.
7. the method that inducing bone mesenchymal stem cell directional according to claim 1 is divided into osteoblast, feature
It is：In the film of the three-dimensional structure, the content of the active matter for promoting osteoblast to adhere to and be proliferated is 0.05wt%
~20wt%.
8. the method that inducing bone mesenchymal stem cell directional according to claim 1 is divided into osteoblast, feature
It is：The active matter for promoting osteoblast to adhere to and be proliferated is collagen, and the collagen is type i collagen, collagen concentration 1
The pH value of~15mg/ml, collagen are 4~6.5.
9. the method that inducing bone mesenchymal stem cell directional according to claim 8 is divided into osteoblast, feature
It is：Collagen concentration described in step b is 7~9mg/ml, and pH value is 5~6.5.
10. the method that inducing bone mesenchymal stem cell directional according to claim 1 is divided into osteoblast, feature
It is：Pre-incubation time described in step e is 4~24 hours.
11. the method that inducing bone mesenchymal stem cell directional according to claim 1 is divided into osteoblast, feature
It is：Osteogenic Induction Medium described in step e, which forms, is mainly：Dexamethasone 10-5~10-2mmol/L, β-glycerine phosphorus
Sour 5~20mmol/L of sodium, 0.02~0.05mmol/L of ascorbic acid.
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