CN106047802A - Material for promoting mesenchymal stem cell osteoblastic differentiation and preparation method and application thereof - Google Patents
Material for promoting mesenchymal stem cell osteoblastic differentiation and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a material for promoting mesenchymal stem cell osteoblastic differentiation and solves the technical problem that electrical characteristics of existing electrical active materials cannot match with natural bone healing process. The material is a thin film using strontium titanate as a substrate and strontium ruthenate as a middle layer and having bismuth ferrite deposited on the surface. The invention further provides and a preparation method and application of the material. The material can be used in the technical field of promoting mesenchymal stem cell osteoblastic differentiation.
Description
Technical field
The present invention relates to promote mescenchymal stem cell Osteoblast Differentiation technical field, be specifically related to a kind of promotion mesenchyme dry thin
Method of born of the same parents' Osteoblast Differentiation and its preparation method and application.
Background technology
Owing to stem cell has height self-renewal capacity and multi-lineage potential, it is that the important kind of regenerative medicine is careful
Born of the same parents.The internal complicated microenvironment of simulation stem cell, regulates and controls stem cell destiny, it is achieved its vitro directed differentiation has become regeneration at present
The study hotspot of medical science.Existing numerous studies confirm that somatomedin, topological structure, elastic modelling quantity etc. can independent or collaborative shadows
Ring stem cell differentiation.And in recent years, the surface charge of biomaterial is increasingly becoming another key factor affecting stem cell behavior.
The potential difference existed between the living tissue intact position of skeleton and damage location, is injury potential.As by electricity
One of them electrode of position meter moves to damage location, and another electrode is in intact position surface, then potentiometric finger be can be observed
Pin deflects, and damage location is negative, and intact position is just.This kind of potential difference, it is possible to promote skeletonization, accelerating union of bone fracture.Cause
This, the injury potential characteristic of osseous tissue, will be for promoting that skeletonization divides due to its pivotal role in bone remoulding and repair process
Change, acceleration bone healing provides a kind of new thinking.
The electricity irritation induction stem cell Osteoblast Differentiation of biomaterial mediation receives significant attention in recent years.Wherein have long-term
The piezoelectric of storage surface electric charge such as Barium metatitanate. (BaTiO3), Lithium metaniobate (LN) etc. have in field of biomedical research and grind in a large number
Studying carefully report, result shows that it has good electroactive and biocompatibility, it is possible to strengthen osteoblastic activity, induced osteogenesis
Differentiation.
But, the polarization intensity of these electroactive materials is low, it is impossible to keep polarization for a long time, it is impossible to mate into the mistake of Bone Defect Repari
Journey.
Summary of the invention
The present invention is contemplated to solve existing electroactive material electrology characteristic cannot mate the technology such as nature bone agglutination
Problem, based on injury potential for the importance of Bone Defect Repari Yu reconstruction, utilizes the bismuth ferrite (BFO) of high polarization intensity to build damage
Potentiometric model, sets up simulation injury potential material promoting mescenchymal stem cell Osteoblast Differentiation and its preparation method and application.
To this end, the present invention provides a kind of material promoting mescenchymal stem cell Osteoblast Differentiation, it is with strontium titanates as substrate,
Ruthenic acid strontium is intermediate layer, the thin film of surface deposition bismuth ferrite.
Preferably, the bismuth ferrite thin film thickness on surface is 5～100nm, and intermediate layer ruthenic acid strontium thickness is 5～100nm, substrate
Strontium titanates thickness is 0.5mm, and surface potential is-30mV～-70mV.
Preferably, bismuth ferrite thin film thickness is 20～30nm, and intermediate layer ruthenic acid strontium thickness is 20～30nm, surface potential
For-65～-70mV.
Present invention simultaneously provides the preparation method of a kind of material promoting mescenchymal stem cell Osteoblast Differentiation, it uses pulse
Laser deposition.
Preferably, in the preparation process of the material of promotion mescenchymal stem cell Osteoblast Differentiation, growth temperature is 300-800 DEG C,
The laser frequency of growth intermediate layer ruthenic acid strontium is 1-5HZ；Laser energy is 60-90mj；Partial pressure of oxygen is 10-20pa；Growth time
For 5-30min；The laser frequency of growing surface bismuth ferrite is 5HZ；Laser energy is 70-90mj；Partial pressure of oxygen is 15-20pa；Raw
Long-time is 5-60min.
The present invention also provides for a kind of material promoting mescenchymal stem cell Osteoblast Differentiation and is promoting mescenchymal stem cell skeletonization
Application in differentiation, it comprises the steps: the electroactive bismuth ferric film material model of (1) preparation simulation injury potential；(2)
Mescenchymal stem cell (MSCs) amplification cultivation: use without supplementing stem cell media between Osteoinductive Factor with money, mesenchyme is dry thin
Cultivating after born of the same parents' recovery, trypsinization is also passed on；(3) mescenchymal stem cell is inoculated on material model: take mescenchymal stem cell inoculation
In having the bismuth ferrite thin film surface of polarized meter surface charge, the mescenchymal stem cell culture medium without Osteoinductive Factor is still used to enter
Row is cultivated；(4) sticking of mescenchymal stem cell is detected with Osteoblast Differentiation.
Preferably, in step (2), in the culture bottle of T75 after derived from bone marrow or the recovery of adipose-derived mescenchymal stem cell
Middle adherent growth, every 3 days with 0.25% trypsinization and pass on.
Preferably, in step (3), take 3-6 and be inoculated in bismuth ferrite surface for mescenchymal stem cell, use without osteogenic induction because of
The culture medium of son, is placed in temperature 37 DEG C, the CO of 5%2Incubator is cultivated.
The present invention utilizes the bismuth ferrite (BFO) of high polarization intensity to promote stem cell Osteoblast Differentiation as simulation injury potential
Study model, it will help promote electroactive embedded material application potential in regenerative medicine, have following technical effect that
(1) present invention passes through pulsed laser deposition, is divided by regulation and control laser frequency, laser energy, growth time and oxygen
The parameters such as pressure, obtain simulating the BFO epitaxial thin film material model of injury potential, and have higher polarization intensity.
(2) present invention promotes mescenchymal stem cell Osteoblast Differentiation by simulation injury potential, solves electroactive material electricity
Learn characteristic and cannot mate the technical problems such as nature bone agglutination, it is provided that be a kind of dry thin not against inducible factor regulation and control mesenchyme
The method of born of the same parents' differentiation.
(3) the electroactive material BFO epitaxial film of the simulation injury potential obtained by the present invention has good with MSCs
Affinity, can promote that MSCs's sticks propagation, promote its Osteoblast Differentiation.
(4) present invention can realize direct inducing mesenchymal stem cell orientation point under conditions of being added without any inducible factor
The purpose changed, safety is good, and controllability is strong.
Below in conjunction with the accompanying drawings the present invention is further detailed.
Accompanying drawing explanation
Fig. 1 is the atomic force microscopy of the BFO epitaxial film simulating injury potential in the embodiment of the present invention 1；
Fig. 2 is the surface potential schematic diagram of the BFO epitaxial film simulating injury potential in the embodiment of the present invention 1；
Fig. 3 is that the external mescenchymal stem cell of the BFO epitaxial film simulating injury potential in the embodiment of the present invention 1 cultivates 3
Hour immunofluorescence photograph；
Fig. 4 is that the BFO epitaxial film simulating injury potential in the embodiment of the present invention 1 promotes mescenchymal stem cell Osteoblast Differentiation
The immunofluorescence dyeing laser co-focusing photo of bone morphogenetic protein 2 (BMP2)；
Fig. 5 is that the BFO epitaxial film simulating injury potential in the embodiment of the present invention 1 promotes mescenchymal stem cell Osteoblast Differentiation
The immunofluorescence dyeing laser co-focusing photo of Runx associated transcription factor 2 (RUNX2).
Detailed description of the invention
The invention provides a kind of method simulating injury potential promotion mescenchymal stem cell Osteoblast Differentiation, below in conjunction with attached
The present invention will be further described with detailed description of the invention for figure.
MSCs cell used in the following example, purchased from Sai Ye bio tech ltd.
Embodiment 1
(1) electroactive bismuth ferrite (BFO) the thin-film material model of preparation simulation injury potential.With strontium titanates (STO) as base
The end, use the BFO epitaxial film of pulsed laser deposition (PLD) preparation simulation injury potential.Growth temperature is 700 DEG C, growth
The laser frequency of intermediate layer ruthenic acid strontium is 2HZ；Laser energy is 70mj；Partial pressure of oxygen is 13pa；Growth time is 20min.Growth
The laser frequency of BFO is 5HZ；Laser energy is 75mj；Partial pressure of oxygen is 18pa；Growth time is 30min.
(2) derived from bone marrow mescenchymal stem cell amplification cultivation: use without supplementing stem cell cultivation between Osteoinductive Factor with money
Base (all buy in match industry by hyclone+100 IU/mL Pen .-Strep of mescenchymal stem cell basal medium+10%
Bio tech ltd, lower same).After MSCs cell recovery in the culture bottle of T75 adherent growth, every 3 days with 0.25%
Trypsinization is also passed on.
(3) induction MSCs is to Osteoblast Differentiation: takes 3-6 and is inoculated in BFO epitaxial film surface for MSCs, uses without osteogenic induction
The culture medium of the factor, is placed in temperature 37 DEG C, the CO of 5%2Incubator is cultivated.
(4) after cultivating 3h, MSCs sticks observation, including cell spreading area and focal adhension differential expression.
(5) MSCs Osteoblast Differentiation detection after 1d is cultivated, including the immunofluorescence dyeing of BMP2, RUNX2.
Being 20～30nm by the BFO film thickness of above step gained, uniformity is good, and crystal grain arrangement is closely and surface is put down
Whole.Intermediate layer ruthenic acid strontium thickness is 20～30nm, and STO substrate thickness is 0.5mm.Surface potential is-65～-70mV.Have good
Biocompatibility, may advantageously facilitate mescenchymal stem cell Osteoblast Differentiation.
Embodiment 2
(1) electroactive material bismuth ferrite (BFO) thin film of preparation simulation injury potential.With STO as substrate, pulse is used to swash
The BFO epitaxial film of Photodeposition (PLD) simulation injury potential.Growth temperature is 300 DEG C, the laser frequency of growth intermediate layer SRO
Rate is 5HZ；Laser energy is 80mj；Partial pressure of oxygen is 15pa；Growth time is 24min.During growth BFO, laser frequency is 5HZ；
Laser energy is 80mj；Partial pressure of oxygen is 15pa；Growth time is 90min.
(2) derived from bone marrow mescenchymal stem cell amplification cultivation: use without supplementing stem cell cultivation between Osteoinductive Factor with money
Base, after MSCs cell recovery in the culture bottle of T75 adherent growth, every 3d with 0.25% trypsinization and pass on.
(3) induction MSCs is to Osteoblast Differentiation: takes 3-6 and is inoculated in the BFO surface of different polarised direction for MSCs, uses without becoming
The culture medium of bone-inducing factor, is placed in temperature 37 DEG C, the CO of 5%2Incubator is cultivated.
(4) after cultivating 3h, MSCs sticks observation, including cell spreading area and focal adhension differential expression.
(5) MSCs Osteoblast Differentiation detection after 1d is cultivated, including the immunofluorescence dyeing of BMP2, RUNX2.
Being 70-80nm by the BFO film thickness of above step gained, uniformity is poor, and crystal grain is arranged relatively in a jumble and table
There is bulky grain in face.Intermediate layer thickness be 70-80nm, STO substrate thickness be 0.5mm, surface potential is-30～-45mV, induction
MSCs Osteoblast Differentiation index BMP2, RUNX2 expression are low.
Embodiment 3
(1) electroactive material bismuth ferrite (BFO) thin film of preparation simulation injury potential.With STO as substrate, pulse is used to swash
The BFO epitaxial film of Photodeposition (PLD) simulation injury potential.Growth temperature is 500 DEG C, the laser frequency of growth intermediate layer SRO
Rate is 5HZ；Laser energy is 95mj；Partial pressure of oxygen is 20pa；Growth time is 2min.During growth BFO, laser frequency is 5HZ；
Laser energy is 105mj；Partial pressure of oxygen is 20pa；Growth time is 10min.
(2) derived from bone marrow mescenchymal stem cell amplification cultivation: use without supplementing stem cell cultivation between Osteoinductive Factor with money
Base, after MSCs cell recovery in the culture bottle of T75 adherent growth, every 3d with 0.25% trypsinization and pass on.
(3) induction MSCs is to Osteoblast Differentiation: takes 3-6 and is inoculated in the BFO surface of different polarised direction for MSCs, uses without becoming
The culture medium of bone-inducing factor, is placed in temperature 37 DEG C, the CO of 5%2Incubator is cultivated.
(4) after cultivating 3h, MSCs sticks observation, including cell spreading area and focal adhension differential expression.
(5) MSCs Osteoblast Differentiation detection after 1d is cultivated, including the immunofluorescence dyeing of BMP2, RUNX2.
Being 5-10nm by the simulation injury potential BFO film thickness of above step gained, uniformity is poor, and compactness is relatively
Low.LSMO or the SRO thickness in intermediate layer be 5-10nm, STO substrate thickness be 0.5mm, surface potential is-45～-60mV, induction
MSCs Osteoblast Differentiation index BMP2, RUNX2 expression are relatively low.
Claims (8)
1. promote a material for mescenchymal stem cell Osteoblast Differentiation, it is characterized in that described material is with strontium titanates as substrate, ruthenium
Acid strontium is intermediate layer, the thin film of surface deposition bismuth ferrite.
The material of promotion mescenchymal stem cell Osteoblast Differentiation the most according to claim 1, it is characterised in that the ferrous acid on surface
Bismuth thin film thickness is 5～100nm, and intermediate layer ruthenic acid strontium thickness is 5～100nm, and substrate strontium titanates thickness is 0.5mm, surface electricity
Gesture is-30mV～-70mV.
The material of promotion mescenchymal stem cell Osteoblast Differentiation the most according to claim 2, it is characterised in that described bismuth ferrite
Film thickness is 20～30nm, and intermediate layer ruthenic acid strontium thickness is 20～30nm, surface potential is-65～-70mV.
4. the preparation method of the material promoting mescenchymal stem cell Osteoblast Differentiation as claimed in claim 1, is characterized in that using
Pulsed laser deposition.
The preparation method of the material of promotion mescenchymal stem cell Osteoblast Differentiation the most according to claim 4, it is characterised in that
In described preparation process, growth temperature is 300-800 DEG C, and the laser frequency of growth intermediate layer ruthenic acid strontium is 1-5HZ；Laser energy
For 60-90mj；Partial pressure of oxygen is 10-20pa；Growth time is 5-30min；The laser frequency of growing surface bismuth ferrite is 5HZ；Swash
Light energy is 70-90mj；Partial pressure of oxygen is 15-20pa；Growth time is 5-60min.
6. the material promoting mescenchymal stem cell Osteoblast Differentiation as claimed in claim 1 is promoting that mescenchymal stem cell skeletonization divides
Application in change, is characterized in that comprising the steps:
(1) the electroactive bismuth ferric film material model of preparation simulation injury potential；
(2) mescenchymal stem cell amplification cultivation: use without supplementing stem cell media between Osteoinductive Factor with money, mesenchyme is dry thin
Cultivating after born of the same parents' recovery, trypsinization is also passed on；
(3) mescenchymal stem cell is inoculated on material model: take mescenchymal stem cell and be inoculated in the ferrous acid with polarized meter surface charge
Bismuth thin film surface, still uses the mescenchymal stem cell culture medium without Osteoinductive Factor to cultivate；
(4) sticking of mescenchymal stem cell is detected with Osteoblast Differentiation.
The material of promotion mescenchymal stem cell Osteoblast Differentiation the most according to claim 6 is promoting mescenchymal stem cell skeletonization
Application in differentiation, it is characterised in that in described step (2), derived from bone marrow or adipose-derived mescenchymal stem cell recovery after in
Adherent growth in the culture bottle of T75, every 3 days with 0.25% trypsinization and pass on.
The material of promotion mescenchymal stem cell Osteoblast Differentiation the most according to claim 6 is promoting mescenchymal stem cell skeletonization
Application in differentiation, it is characterised in that in described step (3), takes 3-6 and is inoculated in bismuth ferrite surface for mescenchymal stem cell, uses
Without the culture medium of Osteoinductive Factor, it is placed in temperature 37 DEG C, the CO of 5%2Incubator is cultivated.
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Cited By (4)
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| CN106893693A (en) * | 2017-03-28 | 2017-06-27 | 周婧 | A kind of method of inducing bone mesenchymal stem cell to osteoblast differentiation |
| CN107469150A (en) * | 2017-08-07 | 2017-12-15 | 北京大学口腔医学院 | A kind of implantation material for forming built-in electric field and preparation method thereof |
| CN115074314A (en) * | 2022-03-28 | 2022-09-20 | 北京大学口腔医学院 | Material for regulating and controlling osteogenic differentiation of stem cells and preparation method and application thereof |
| WO2024098285A1 (en) * | 2022-11-09 | 2024-05-16 | 深圳先进技术研究院 | Exosome program-controlled tissue repair material and preparation method therefor |
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| CN107469150A (en) * | 2017-08-07 | 2017-12-15 | 北京大学口腔医学院 | A kind of implantation material for forming built-in electric field and preparation method thereof |
| CN115074314A (en) * | 2022-03-28 | 2022-09-20 | 北京大学口腔医学院 | Material for regulating and controlling osteogenic differentiation of stem cells and preparation method and application thereof |
| WO2024098285A1 (en) * | 2022-11-09 | 2024-05-16 | 深圳先进技术研究院 | Exosome program-controlled tissue repair material and preparation method therefor |
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