CN103789257B - Directional differentiation induction method for mesenchymal stem cells - Google Patents
Directional differentiation induction method for mesenchymal stem cells Download PDFInfo
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- CN103789257B CN103789257B CN201410011155.5A CN201410011155A CN103789257B CN 103789257 B CN103789257 B CN 103789257B CN 201410011155 A CN201410011155 A CN 201410011155A CN 103789257 B CN103789257 B CN 103789257B
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Abstract
The invention discloses a method for preparing a titanium dioxide (TiO2) nano-tube with a micro-nano multi-level structure to regulate the directional differentiation of mesenchymal stem cells into smooth-muscle-like cells. The method comprises the following steps: firstly, preparing a TiO2 nano-tube micro-channel structure by the combination of an ultraviolet patterning lithography and an anodic oxidation technology; secondly, directly inducing the directional differentiation of the mesenchymal stem cells into the smooth-muscle-like cells on the surface of the prepared TiO2 nano-tube micro-channel structure by using a frequent passage and medium change method under the condition of no addition of any biochemical inducing factors. According to the method, the TiO2 nano-tube with the micro-nano multi-level structure is prepared on the surface of Ti, and the aim of directly inducing the mesenchymal stem cells is achieved in combination with the frequent passage and medium change method under the condition of no addition of any biochemical inducing factors.
Description
Technical field
The present invention relates to and utilize litho pattern combine with technique anodic oxidation producing nanotubes, prepare a kind of patterning material interface of nanotube of micro-nano multi-level structure, and utilize a kind of technology of the method induced dry-cell directed differentiation on this material interface of changing liquid that frequently goes down to posterity, belong to Surface Engineering of Biomaterials and organizational project crossing domain.
Background technology
Optical etching technology has a very wide range of applications in the field such as electronics, chemical industry, organizational engineering particularly biomedical engineering field also play very important effect, this is because the material surface topological framework of micrometer/nanometer rank all plays certain impact to the adhesion of cell, propagation, differentiation, apoptosis.Multiple dimensioned micro-nano compound structure plays important, sometimes or even conclusive effect in bioelectric interface.The internal surface that original position bioluminescence imaging technology discloses real blood vessels is exactly the very typical multiple dimensioned micro-nano compound structure of one: along submicron-scale groove and the nano level bag-like projection of flute surfaces of blood flow direction.Pointed out the very low power with nanostructure can the growth orientation of unusual good control cell, playing the arrangement mode that simulation people in-vivo tissue is natural, be imitation biochemistry important reference.The present invention prepares a kind of brand-new titanium dioxide (TiO with reference to optical etching technology
2) the surface tissue form of nanotube very low power, and utilize this material interface directional induction mescenchymal stem cell (MSCs) to be divided into class unstriated muscle.
Titanium and titanium alloys has a very wide range of applications on biomedical embedded material, comprises, bone and osteoarticular displacement apparatus, dental material, artificial blood vessel bracket, prosthetic heart valve and jaw face and cranium face support implantable material etc.But titanium is as a kind of inert material, the cytocompatibility sexual needs after implant into body improve further, and TiO
2nanotube has unique structure and excellent character, as high surface energy, bigger serface, Superhydrophilic matter etc., is therefore widely used in above such as biosensor, solar cell, photochemical catalysis, photoelectrolysis and bio-medical material etc.Sizable result of study shows, TiO
2the surface of nanostructure can change by active and effective promotion cell behavior, such as promotes that the calcium-phosphorus ratio of material surface accelerates to be formed, strengthens the adhesion of osteocyte, propagation and differentiation etc.And by TiO
2nanotube carries out in the process studied as Implantable Medical Device, had a lot of cytology evaluation, at research TiO
2after nanotube loading BMP-2 cell inducible factors, directional induction differentiation of stem cells is that scleroblast has had some achievements in research.But, will TiO be possessed
2there is not been reported in the research of the very low power applying biological Material Field of nanostructure; TiO will be possessed
2the very low power of nanostructure is applied to the research of stem cell directional differentiation, and also there is not been reported.
Current preparation TiO
2the method of nanotube mainly contains following several, auxiliary template method, electrochemistry anodic oxidation, and hydrothermal synthesis method etc.Wherein the method for electrochemical anodic oxidation is because of high-sequential in the Nanotube alignment mode of preparation, and consistence is high, thus is widely used.Nanotube specification prepared by this method, as caliber, the high length being subject to different electrolytes, applied voltage, pH value height and anodizing time of pipe controls.The titania nanotube generated through anodic oxidation is non-crystalline state, is also the TiO of amorphous state
2nanotube, and after Overheating Treatment TiO
2nanotube forms the multiphase of rutile and anatase octahedrite.TiO
2nanotube is preparation technology be mature on the whole.The preparation technology of once oxidation pattern has been reported research.But, prepare the multiple dimensioned micro-nano compound structure of titanium dioxide in conjunction with photoetching technique and two-step anodization technology---directly allow nanotube be very low power distribution technique there is not been reported.
Stem cell is since being found, move under water because of its potential many differentiation and become the only choosing of organizational engineering seed cell with good multiplication capacity, and mescenchymal stem cell (MSCs) is as a kind of wide material sources, stem cell easily of drawing materials also becomes the focus of seed cell research.In the work of research material inducing mesenchymal stem cell (MSCs) directed differentiation, the method that tradition adopts is cultivated for a long time the stem cell being directly planted in material surface, replacement operation is not carried out in culturing process, and need to add the biochemical induction factor, and this research employing is that a kind of frequently going down to posterity changes the training mode of liquid, at once digest after cell is paved with sample surfaces, overhang and be again inoculated into brand-new material surface, 10-20 culture so repeatedly, until identifying till differentiation of stem cells is class smooth muscle cell, time probably continued about 4-8 week.Under this condition not adding any biochemical induction factor, only with the research of the inducing action of frequent method strengthening groove structure titania nanotube of regenerating, there is not been reported.
Summary of the invention
In view of studying TiO at present
2generally need adding of the biochemical induction factor in the process of nanotube directional induction stem cell, and training method is do not go down to posterity formula short-term or long-term cultivation, there is no the direct induction factor that real reference material is induced stem cell.Main purpose of the present invention is to provide a kind of novel method of directed differentiation induction of mescenchymal stem cell, utilizes UV-light to etch technological incorporation anodizing technology and prepares a kind of TiO
2nanotube very low power structure, and combine and frequently go down to posterity and change the cultivation of liquid, under the prerequisite not adding the biochemical induction factor, the direct differentiation of stem cells of nanotube very low power structure, and finally obtain class smooth muscle cell.
The present invention is realized mainly through following technological line.
A kind of directed differentiation induction method of mescenchymal stem cell, utilizing micro-nano multi-level structure titania nanotube to regulate and control mescenchymal stem cell directed differentiation is class smooth muscle cell, utilize UV-light to etch technological incorporation anodizing technology and prepare titania nanotube very low power structure, and combine and frequently go down to posterity and change the cultural method of liquid, under the prerequisite not adding the biochemical induction factor, direct differentiation of stem cells in described very low power structure, and final acquisition class smooth muscle cell, comprise following steps:
1) with TiO
2the very low power preparation of nano tube structure
Use UV-light etching technological incorporation anodizing technology to prepare micrographics controlled in order and nano tube structure at medical embedded metallic titanium surface simultaneously---micro-nano multi-level structure surface, described micrographics is that long strip shape ditch/ridge is interspersed structure, ditch/ridge width is respectively 5-25 μm/5-25 μm, nanotube caliber is 30-100nm, thickness of pipe is about 10-50nm, pipe range is 300-500nm, and groove drop is 300-500nm;
2) frequently go down to posterity and change liquid Induction of committed differentiation MSCs
MSCs is directly incubated at 1) gained micro-nano multi-level structure surface, under the prerequisite not adding any biochemical induction factor, rely on TiO
2micro-nano multi-level structure itself is carried out cell and is changed liquid cultivation, and final acquisition class smooth muscle cell, preparation process is: by the MSCs cell of primary extraction with 0.5 × 10
4-2 × 10
4the density of individual/ml directly overhangs plantation in the container of titania nanotube having micro-nano multi-level structure; MSCs substratum be without any biochemical induction factor add 85% the foetal calf serum of DMEM basic medium+15%; Change liquid every three days once, cell needs after being paved with sample surfaces to go down to posterity again, so repeatedly 4-8 week, frequently goes down to posterity change liquid cultivation through 10-20 time; Collect sample surfaces cell and obtain target product class smooth muscle cell.
The preparatory phase on described micro-nano multi-level structure surface comprises the steps:
A. the preparation flow of litho pattern:
A) titanium substrate is prepared into sizeable size and by surface finish polishing, for subsequent use after cleaning-drying;
B) take out clean and after drying treatment titanium substrate, use sol evenning machine that positive photoresist is evenly coated in titanium substrate surface, put into baking oven 90-120 DEG C, post bake 10m-30min;
C) lithography machine adds mask plate and carry out uv-exposure operation, light intensity is 16 milliwatts/cm
2, expose 10 seconds, λ=365nm;
D) then carry out the region be mapped to by ultraviolet lighting of developing, photoresist material dissolves, and sample surfaces is then stayed in the region that mask plate covers at once, and the titanium substrate after development after cleaning exposure, just obtains the titanium substrate of photoetching offset plate figure;
E) dry 10-30min after putting into baking oven 90-120 DEG C again to use for next step;
B. the flow process of titania nanotube micrographics is prepared by two-step anodization:
A) configure electrolytic solution, use solute is Neutral ammonium fluoride, and additive is sodium-chlor and glycerol, and remaining solvent is distilled water;
B) jointed anode oxidation unit, using the titanium substrate of coating photoresist material that obtains in step A as positive level, graphite flake, as negative electrode, carries out the process of electrochemistry anode oxidation, can generate the film of the consistent titania nanotube of a kind of uniform diameter at titanium substrate surface;
C) the titanium substrate after an anodic oxidation treatment is immersed the photoresist material of eccysis titanium substrate surface in acetone soln;
D) and sample is immersed in supersound process in glycerin solution, by B-b) anodic oxidation obtains in step the whole ultrasonic vibration of titania nanotube interrupts, and disposes;
E) carry out secondary oxidation, method is with step B-b);
The titania nanotube of the micro-nano structure f) obtained respectively cleans at least three times with dehydrated alcohol, deionized water successively, and carries out oven dry 60 DEG C process;
G) sample that obtains after f) process is carried out 500 ± 50 DEG C, clean again after thermal treatment in 3 ± 1 hours, and dry and preserve.
Frequently go down to posterity the cultural method changing liquid time, the sterilising treatment of sample adopts UV sterilization, is directly seeded in sample surfaces after MSCs original cuiture; Go down to posterity container used and titania nanotube very low power sample time need to use and do not cultivate the titania nanotube very low power body structure surface of stem cell, to ensure that material surface has the influence of a continual induction to former generation cell; When going down to posterity, the planting density of cell remains unchanged.
MSCs cell obtains and adopts primary extraction MSCs to take Graded Density method to carry out.
When cell changes liquid cultivation, change the pancreatin that liquid digestion uses 0.25mg/ml, culture condition is 37 DEG C, 5%CO
2moist environment.
The present invention finally achieves the Induction of committed differentiation to mescenchymal stem cell, and the development for titanium-based metal surface biological, tissue engineering technique provides a kind of more feasible method.
Accompanying drawing explanation
Below in conjunction with drawings and Examples, method of the present invention is described in further detail.
Fig. 1 is with TiO in the inventive method
2each step schematic diagram prepared by the very low power of nano tube structure.
Fig. 2 is the scanning electron microscope (SEM) photograph of sample surface morphology.(a) TiO
2for not through sample prepared by nanotube and very low power; B () NANO is for only to have prepared TiO
2the sample of nanotube; C () NANO-GRO is through once anodised very low power sample; D () NANO-TOPO is anodised with TiO through twice
2the very low power sample of nano tube structure.
Fig. 3 frequently changes each step schematic diagram of liquid method induction MSCs directed differentiation in the inventive method.
Fig. 4 is after sample surfaces MSCs directional induction is cultivated, cell surface specific marker molecule alpha-SMA identified by immunofluorescence and nucleus DAPI fluorescent dye result and fitted figure.(a) TiO
2for not through sample prepared by nanotube and very low power; B () NANO is for only to have prepared TiO
2the sample of nanotube; C () NANO-GRO is through once anodised very low power sample; D () NANO-TOPO is anodised with TiO through twice
2the very low power sample of nano tube structure.
Fig. 5 is after sample surfaces MSCs directional induction is cultivated, cell surface specific marker molecule CNN-1 identified by immunofluorescence and nucleus DAPI fluorescent dye result and fitted figure.(a) TiO
2for not through sample prepared by nanotube and very low power; B () NANO is for only to have prepared TiO
2the sample of nanotube; C () NANO-GRO is through once anodised very low power sample; D () NANO-TOPO is anodised with TiO through twice
2the very low power sample of nano tube structure.
Embodiment
When reality is implemented, ultraviolet photolithographic pattern specimens preparation process is as follows:
1. the sample surfaces coating photoresist material by polishing and after cleaning, drying, after preliminary drying, carries out mask and hides ultraviolet lighting, the sample after exposure is carried out development treatment, carries out post-drying, obtain pattern specimens, treat further process after development;
2. described in sample be titanium substrate, thickness is 50-1000 μm, and purging method is, acetone, dehydrated alcohol, distilled water clean three times successively;
3. the mask described in can be selected arbitrarily as required, and dimension of picture groove selects 5-25/5-25 μm;
4. the pre-bake temperature described in is 90-120 DEG C, 10-30 minute;
5. the photoresist material described in adopts the method for sol evenning machine coating, and sol evenning machine arranges 500r/min at a slow speed, 18 seconds; Quick 3000r/min, 60 seconds; Photoresist material thickness is about 600-800nm;
6. the preliminary drying temperature described in is 90-120 DEG C, 10-30 minute;
7. the time shutter described in is set to 10 seconds;
8. the photographic developer described in is the NaOH aqueous solution of 0.5wt%, development time 10 seconds;
9. the rear baking temperature described in is 90-120 DEG C, 10-30 minute.
The preparation process of twice anodic oxidation titania nanotube very low power is as follows:
1. the patterned sample prepared is carried out first time anodic oxidation, because the photoresist material of sample surfaces exists, the therefore TiO of Primary Oxidation
2the place production that nanotube is only applying without photoresist material, having the ground of photoresist material to put then can not producing nanotubes; By the titania nanotube sample generated for the first time, carry out ultrasonication, object removes the nanotube generated, and wash the photoresist material of sample surfaces; By removing the sample after photoresist material and carry out secondary oxidation after ultrasonication, generate the TiO of patterning structure
2nanotube; The sample obtained being carried out cleaning postheat treatment preserves stand-by;
2. the anode oxidation electrolytic solution described in is V (glycerol): V (H
2the NH of O)=4:1,0.5wt%
4the organic system of F, 0.06Mol NaCl, and the electrolytic solution of twice oxidation is identical; Anodizing time is all set to 2-3 hour, voltage 20V;
3. the ultrasonic vibration of the employing described in removes the frequency 30-50KHz of nanotube, and power is 300-450W, and the time is about 1 hour;
4. the nanotube caliber generated after the secondary oxidation described in is at 30-100nm, and thickness of pipe is about 10-50nm, and pipe range is about 300-500nm, and groove drop is 300-500nm;
5. the heat treating method described in adopts vacuum sintering furnace heat treating method, temperature setting 500 ± 50 DEG C, 3 ± 1 hours time length, obtains the titania nanotube of rutile and anatase octahedrite mixing crystalline structure;
6. the sample clean method described in is dehydrated alcohol, and deionized water respectively cleans at least three times.
Frequently going down to posterity and changing liquid is that material interface is continual induces differentiation of stem cells in order to impel, by the mescenchymal stem cell MSCs of primary extraction, uniform plantation is to the titania nanotube surface of this very low power structure, be paved with after cell until sample surfaces, cell 0.25mg/ml tryptic digestion got off, plants brand-new orifice plate and sample surfaces by initial density, so repeatedly, continue 4-8 week, the cell finally obtained is identified, obtain the cell after breaking up; Described cell culture medium is the foetal calf serum of the DMEM basic medium+15% of 85%; Described sample before repopulating cell through UV sterilising treatment; Described cell initial seeding density is 0.5 × 10
4-2 × 10
4individual/ml; The described method of changing liquid that frequently goes down to posterity is carried out under the state that cell is paved with completely on sample, and cell density when going down to posterity keeps identical with initial density 0.5 × 10
4-2 × 10
4individual/ml.
Embodiment one
See Fig. 1 and Fig. 3, the first embodiment of the present invention, preparation micro-nano multi-level structure titania nanotube, to regulate and control mescenchymal stem cell directed differentiation for class smooth muscle cell, the steps include:
Sample preparation: titanium substrate is prepared into sizeable size and by surface finish polishing, for subsequent use after cleaning-drying; Take out clean and after drying treatment titanium substrate, use sol evenning machine that positive photoresist is evenly coated in titanium substrate surface, put into 90 DEG C, baking oven, post bake 30min; Carry out uv-exposure operation, light intensity is 16 milliwatts/cm
2, expose 10 seconds, λ=365nm; Rear horse back carries out the region be mapped to by ultraviolet lighting of developing, and photoresist material dissolves, and sample surfaces is then stayed in the region that mask plate covers, and the titanium substrate after development after cleaning exposure, just obtains the titanium substrate of photoetching offset plate figure; 30min is dried after putting into 90 DEG C, baking oven again, stand-by; By twice anodic oxidation, at gained micrographics surface preparation TiO
2nanotube, obtains through 500 DEG C of thermal treatments and has TiO for 2 hours
2the very low power of nano tube structure;
Utilizing frequently goes down to posterity changes liquid method and has TiO
2the microgroove surface inducing mesenchymal stem cell directed differentiation of nano tube structure: by the sample after sterilising treatment, is put into without in the processed blank container of any bag.By the MSCs cell of primary extraction with 2 × 10
4the density of individual/ml directly overhangs plantation to the TiO having micro-nano multi-level structure
2in the container of nanotube; Change liquid every three days once, cell need with 2 × 10 after being paved with sample surfaces
4the density of individual/ml goes down to posterity again; So 8 weeks repeatedly, frequently go down to posterity through 12 times and change liquid cultivation; Collect sample surfaces cell and carry out a series of signs such as flow cytometer/Fluorescence Identification.And determine that obtaining cell is class smooth muscle cell eventually through Fluorescence Identification.
Embodiment two
Prepare micro-nano multi-level structure titania nanotube to regulate and control the method for mescenchymal stem cell directed differentiation for class smooth muscle cell, the steps include:
Sample preparation: titanium substrate is prepared into sizeable size and by surface finish polishing, for subsequent use after cleaning-drying; Take out clean and after drying treatment titanium substrate, use sol evenning machine that positive photoresist is evenly coated in titanium substrate surface, put into 120 DEG C, baking oven, post bake 10min; Carry out uv-exposure operation, light intensity is 16 milliwatts/cm
2, expose 10 seconds, λ=365nm; Rear horse back carries out the region be mapped to by ultraviolet lighting of developing, and photoresist material dissolves, and sample surfaces is then stayed in the region that mask plate covers, and the titanium substrate after development after cleaning exposure, just obtains the titanium substrate of photoetching offset plate figure; 10min is dried after putting into 120 DEG C, baking oven again, stand-by; By twice anodic oxidation, at gained micrographics surface preparation TiO
2nanotube, obtains through 450 DEG C of thermal treatments and has TiO for 3 hours
2the very low power of nano tube structure;
Utilizing frequently goes down to posterity changes liquid method and has TiO
2the microgroove surface inducing mesenchymal stem cell directed differentiation of nano tube structure: by the sample after sterilising treatment, is put into without in the processed blank container of any bag.By the MSCs cell of primary extraction with 0.5 × 10
4the density of individual/ml directly overhangs plantation to the TiO having micro-nano multi-level structure
2in the container of nanotube; Change liquid every three days once, cell need with 0.5 × 10 after being paved with sample surfaces
4the density of individual/ml goes down to posterity again; So 8 weeks repeatedly, frequently go down to posterity through 20 times and change liquid cultivation; Collect sample surfaces cell and carry out a series of signs such as flow cytometer/Fluorescence Identification.And determine that obtaining cell is class smooth muscle cell eventually through Fluorescence Identification.
Embodiment three
Prepare micro-nano multi-level structure titania nanotube to regulate and control the method for mescenchymal stem cell directed differentiation for class smooth muscle cell, the steps include:
Sample preparation: titanium substrate is prepared into sizeable size and by surface finish polishing, for subsequent use after cleaning-drying; Take out clean and after drying treatment titanium substrate, use sol evenning machine that positive photoresist is evenly coated in titanium substrate surface, put into 105 DEG C, baking oven, post bake 20min; Carry out uv-exposure operation, light intensity is 16 milliwatts/cm
2, expose 10 seconds, λ=365nm; Rear horse back carries out the region be mapped to by ultraviolet lighting of developing, and photoresist material dissolves, and sample surfaces is then stayed in the region that mask plate covers, and the titanium substrate after development after cleaning exposure, just obtains the titanium substrate of photoetching offset plate figure; 20min is dried after putting into 105 DEG C, baking oven again, stand-by; By twice anodic oxidation, at gained micrographics surface preparation TiO
2nanotube, obtains through 450 DEG C of thermal treatments and has TiO for 3 hours
2the very low power of nano tube structure;
Utilizing frequently goes down to posterity changes liquid method and has TiO
2the microgroove surface inducing mesenchymal stem cell directed differentiation of nano tube structure: by the sample after sterilising treatment, is put into without in the processed blank container of any bag.By the MSCs cell of primary extraction with 1 × 10
4the density of individual/ml directly overhangs plantation to the TiO having micro-nano multi-level structure
2in the container of nanotube; Change liquid every three days once, cell need with 1 × 10 after being paved with sample surfaces
4the density of individual/ml goes down to posterity again; So 6 weeks repeatedly, frequently go down to posterity through 16 times and change liquid cultivation; Collect sample surfaces cell and carry out a series of signs such as flow cytometer/Fluorescence Identification.And determine that obtaining cell is class smooth muscle cell eventually through Fluorescence Identification.
Claims (5)
1. the directed differentiation induction method of a mescenchymal stem cell, utilizing micro-nano multi-level structure titania nanotube to regulate and control mescenchymal stem cell directed differentiation is class smooth muscle cell, utilize UV-light to etch technological incorporation anodizing technology and prepare titania nanotube very low power structure, and combine and frequently go down to posterity and change the cultural method of liquid, under the prerequisite not adding the biochemical induction factor, direct differentiation of stem cells in described very low power structure, and final acquisition class smooth muscle cell, comprise following steps:
1) with TiO
2the very low power preparation of nano tube structure
Use UV-light etching technological incorporation anodizing technology to prepare micrographics controlled in order and nano tube structure at medical embedded metallic titanium surface simultaneously---micro-nano multi-level structure surface, described micrographics is that long strip shape ditch/ridge is interspersed structure, ditch/ridge width is respectively 5-25 μm/5-25 μm, nanotube caliber is 30-100nm, thickness of pipe is about 10-50nm, pipe range is 300-500nm, and groove drop is 300-500nm;
2) frequently go down to posterity and change liquid Induction of committed differentiation MSCs
MSCs is directly incubated at 1) gained micro-nano multi-level structure surface, under the prerequisite not adding any biochemical induction factor, rely on TiO
2micro-nano multi-level structure itself is carried out cell and is changed liquid cultivation, and final acquisition class smooth muscle cell, preparation process is: by the MSCs cell of primary extraction with 0.5 × 10
4-2 × 10
4the density of individual/ml directly overhangs plantation in the container of titania nanotube having micro-nano multi-level structure; MSCs substratum be without any biochemical induction factor add 85% the foetal calf serum of DMEM basic medium+15%; Change liquid every three days once, cell needs after being paved with sample surfaces to go down to posterity again, so repeatedly 4-8 week, frequently goes down to posterity and changes liquid cultivation, collect sample surfaces cell and obtain target product class smooth muscle cell through 10-20 time.
2. the directed differentiation induction method of mescenchymal stem cell according to claim 1, it is characterized in that, the preparation on described micro-nano multi-level structure surface comprises the steps:
A. the preparation flow of litho pattern:
A) titanium substrate is prepared into sizeable size and by surface finish polishing, for subsequent use after cleaning-drying;
B) take out clean and after drying treatment titanium substrate, use sol evenning machine that positive photoresist is evenly coated in titanium substrate surface, put into baking oven 90-120 DEG C, post bake 10m-30min;
C) lithography machine adds mask plate and carry out uv-exposure operation, light intensity is 16 milliwatts/cm
2, expose 10 seconds, λ=365nm;
D) then carry out the region be mapped to by ultraviolet lighting of developing, photoresist material dissolves, and sample surfaces is then stayed in the region that mask plate covers at once, and the titanium substrate after development after cleaning exposure, just obtains the titanium substrate of photoetching offset plate figure;
E) dry 10-30min after putting into baking oven 90-120 DEG C again to use for next step;
B. the flow process of titania nanotube micrographics is prepared by two-step anodization:
A) configure electrolytic solution, use solute is Neutral ammonium fluoride, and additive is sodium-chlor and glycerol, and remaining solvent is distilled water;
B) jointed anode oxidation unit, using the titanium substrate of coating photoresist material that obtains in step A as positive level, graphite flake, as negative electrode, carries out the process of electrochemistry anode oxidation, can generate the film of the consistent titania nanotube of a kind of uniform diameter at titanium substrate surface;
C) the titanium substrate after an anodic oxidation treatment is immersed the photoresist material of eccysis titanium substrate surface in acetone soln;
D) and sample is immersed in supersound process in glycerin solution, by B-b) anodic oxidation obtains in step the whole ultrasonic vibration of titania nanotube interrupts, and disposes;
E) carry out secondary oxidation, method is with step B-b);
The titania nanotube of the micro-nano structure f) obtained respectively cleans at least three times with dehydrated alcohol, deionized water successively, and carries out oven dry 60 DEG C process;
G) sample that obtains after f) process is carried out 500 ± 50 DEG C, clean again after thermal treatment in 3 ± 1 hours, and dry and preserve.
3. the directed differentiation induction method of mescenchymal stem cell according to claim 1, it is characterized in that, in class smooth muscle cell procurement process, primary extraction MSCs takes Graded Density method to carry out.
4. the directed differentiation induction method of mescenchymal stem cell according to claim 1, is characterized in that, in class smooth muscle cell procurement process, cell changes liquid when cultivating, and change the pancreatin that liquid digestion uses 0.25mg/ml, culture condition is 37 DEG C, 5%CO
2moist environment.
5. the directed differentiation induction method of the mescenchymal stem cell according to claim 1 or 2, is characterized in that, frequently go down to posterity the cultural method changing liquid time, the sterilising treatment of sample adopts UV sterilization, is directly seeded in sample surfaces after MSCs original cuiture; Go down to posterity container used and titania nanotube very low power sample time need to use and do not cultivate the titania nanotube very low power body structure surface of stem cell, to ensure that material surface has the influence of a continual induction to former generation cell; When going down to posterity, the planting density of cell remains unchanged.
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