CN106047802B - A kind of material and its preparation method and application promoting mescenchymal stem cell Osteoblast Differentiation - Google Patents
A kind of material and its preparation method and application promoting mescenchymal stem cell Osteoblast Differentiation Download PDFInfo
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Abstract
The present invention relates to a kind of materials and its preparation method and application for promoting mescenchymal stem cell Osteoblast Differentiation, which solve existing electroactive material electrology characteristics can not match the technical problems such as nature bone agglutination, promoting the material of mescenchymal stem cell Osteoblast Differentiation in the present invention is using strontium titanates as substrate, ruthenic acid strontium is middle layer, and surface deposits the film of bismuth ferrite.Invention also provides preparation methods and application.The present invention can be used for promoting mescenchymal stem cell Osteoblast Differentiation technical field.
Description
Technical field
The present invention relates to promote mescenchymal stem cell Osteoblast Differentiation technical field, and in particular to a kind of promotion mesenchyma is dry thin
The method and its preparation method and application of born of the same parents' Osteoblast Differentiation.
Background technique
It is that important kind of regenerative medicine is careful since stem cell has height self-renewal capacity and multi-lineage potential
Born of the same parents.The internal complicated microenvironment for simulating stem cell, regulates and controls stem cell destiny, realizes that its vitro directed differentiation has become current regeneration
The research hotspot of medicine.Existing numerous studies confirm that growth factor, topological structure, elasticity modulus etc. can independent or collaboration shadows
Ring stem cell differentiation.And in recent years, the surface charge of biomaterial is increasingly becoming another key factor for influencing stem cell behavior.
Existing potential difference, as demarcation potential between the living tissue intact position of bone and damage location.It such as will be electric
One of electrode of position meter moves to damage location, and another electrode is in intact position surface, then potentiometric finger can be observed
Needle deflects, and damage location is negative, and intact position is positive.Such potential difference can promote skeletonization, accelerating union of bone fracture.Cause
This, the demarcation potential characteristic of bone tissue will be promotion skeletonization point due to its key effect in bone remoulding and repair process
Change, bone healing is accelerated to provide a kind of new thinking.
The electro photoluminescence induction stem cell Osteoblast Differentiation that biomaterial mediates receives significant attention in recent years.Wherein have long-term
The piezoelectric material of storage surface charge such as barium titanate (BaTiO3), lithium niobate (LN) etc. have in field of biomedical research largely to be ground
Study carefully report, it can enhance the activity of osteoblast, induced osteogenesis with good electroactive and biocompatibility as the result is shown
Differentiation.
However, the polarization intensity of these electroactive materials is low, it cannot keep polarizing for a long time, the mistake of Bone Defect Repari can not be matched into
Journey.
Summary of the invention
The present invention is exactly that can not match the technologies such as nature bone agglutination to solve existing electroactive material electrology characteristic
Problem constructs damage using the bismuth ferrite (BFO) of high polarization intensity based on demarcation potential for the importance of Bone Defect Repari and reconstruction
Potentiometric model establishes the material and its preparation method and application that simulation demarcation potential promotes mescenchymal stem cell Osteoblast Differentiation.
For this purpose, the present invention provide it is a kind of promote mescenchymal stem cell Osteoblast Differentiation material, be using strontium titanates as substrate,
Ruthenic acid strontium is middle layer, and surface deposits the film of bismuth ferrite.Bismuth ferrite thin film is with a thickness of 20~30nm, middle layer ruthenic acid strontium thickness
For 20~30nm, surface potential is -65~-70mV.
Present invention simultaneously provides a kind of preparation methods of material for promoting mescenchymal stem cell Osteoblast Differentiation, use pulse
Laser deposition.
Preferably, promoting growth temperature in the preparation process of the material of mescenchymal stem cell Osteoblast Differentiation is 300-800 DEG C,
The laser frequency for growing middle 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；It is raw
It is for a long time 5-60min.
The present invention also provides a kind of materials for promoting mescenchymal stem cell Osteoblast Differentiation to promote mescenchymal stem cell skeletonization
Application in differentiation comprising following steps: (1) the electroactive bismuth ferric film material model of preparation simulation demarcation potential；(2)
Mescenchymal stem cell (MSCs) amplification cultivation: using without stem cell media is supplemented between Osteoinductive Factor with money, mesenchyma is dry thin
It is cultivated after born of the same parents' recovery, pancreatin is digested and passed on；(3) mescenchymal stem cell is inoculated on material model: mescenchymal stem cell being taken to be inoculated with
In have polarization surface charge bismuth ferrite thin film surface, still using the mescenchymal stem cell culture medium without Osteoinductive Factor into
Row culture；(4) sticking for 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 is digested and is passed on 0.25% pancreatin for every 3 days.
Preferably, in step (3), 3-6 is taken to be inoculated in bismuth ferrite surface for mescenchymal stem cell, using no osteogenic induction because
The culture medium of son, is placed in 37 DEG C of temperature, 5% CO2It is cultivated in incubator.
The present invention promotes stem cell Osteoblast Differentiation using the bismuth ferrite (BFO) of high polarization intensity as simulation demarcation potential
Research model, it will help promote the application potential of electroactive implantation material in regenerative medicine, have following technical effect that
(1) present invention passes through regulation laser frequency, laser energy, growth time and oxygen by pulsed laser deposition
The parameters such as pressure obtain the BFO epitaxial thin film material model of simulation demarcation potential, and polarization intensity with higher.
(2) present invention promotes mescenchymal stem cell Osteoblast Differentiation by simulation demarcation potential, solves electroactive material electricity
The technical problems such as nature bone agglutination can not be matched by learning characteristic, be provided a kind of dry thin not against inducible factor regulation mesenchyma
The method of born of the same parents' differentiation.
(3) the electroactive material BFO epitaxial film of the simulation demarcation potential obtained by the present invention has good with MSCs
Compatibility, can promote MSCs sticks proliferation, promotes its Osteoblast Differentiation.
(4) direct inducing mesenchymal stem cell orientation point can be achieved in the present invention under conditions of being added without any inducible factor
The purpose of change, safety is good, and controllability is strong.
The present invention is further detailed with reference to the accompanying drawing.
Detailed description of the invention
Fig. 1 is the atomic force microscopy that the BFO epitaxial film of demarcation potential is simulated in the embodiment of the present invention 1；
Fig. 2 is the surface potential schematic diagram that the BFO epitaxial film of demarcation potential is simulated in the embodiment of the present invention 1；
Fig. 3 is the external mescenchymal stem cell culture 3 that the BFO epitaxial film of demarcation potential is simulated in the embodiment of the present invention 1
The immunofluorescence photograph of hour；
Fig. 4 is the BFO epitaxial film promotion mescenchymal stem cell Osteoblast Differentiation that demarcation potential is simulated in the embodiment of the present invention 1
Bone morphogenetic protein 2 (BMP2) immunofluorescence dyeing laser co-focusing photo；
Fig. 5 is the BFO epitaxial film promotion mescenchymal stem cell Osteoblast Differentiation that demarcation potential is simulated in the embodiment of the present invention 1
Runx associated transcription factor 2 (RUNX2) immunofluorescence dyeing laser co-focusing photo.
Specific embodiment
The present invention provides a kind of methods that simulation demarcation potential promotes mescenchymal stem cell Osteoblast Differentiation, below with reference to attached
The present invention will be further described with specific embodiment for figure.
MSCs cell used in the following example is purchased from Sai Ye Biotechnology Co., Ltd.
Embodiment 1
(1) electroactive bismuth ferrite (BFO) thin-film material model of preparation simulation demarcation potential.With strontium titanates (STO) for base
Bottom prepares the BFO epitaxial film of simulation demarcation potential using pulsed laser deposition (PLD).Growth temperature is 700 DEG C, growth
The laser frequency of middle 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 is cultivated without stem cell is supplemented between Osteoinductive Factor with money
(fetal calf serum+100IU/mL Pen .- Strep of mescenchymal stem cell basal medium+10%, purchase are raw in match industry for base
Object Science and Technology Ltd., similarly hereinafter).After MSCs cell recovery in the culture bottle of T75 adherent growth, every 3 days with 0.25% pancreas
Enzymic digestion is simultaneously passed on.
(3) induction MSCs is to Osteoblast Differentiation: taking 3-6 to be inoculated in BFO epitaxial film surface for MSCs, using no osteogenic induction
The culture medium of the factor is placed in 37 DEG C of temperature, 5% CO2It is cultivated in incubator.
(4) MSCs sticks observation, including cell spreading area and focal adhension differential expression after cultivating 3h.
(5) MSCs Osteoblast Differentiation detects after cultivating 1d, the immunofluorescence dyeing including BMP2, RUNX2.
It is 20~30nm by the resulting BFO film thickness of above step, uniformity is good, and crystal grain arrangement is close and surface is flat
It is whole.For middle layer ruthenic acid strontium with a thickness of 20~30nm, 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) film of preparation simulation demarcation potential.Using STO as substrate, swashed using pulse
The BFO epitaxial film of Photodeposition (PLD) simulation demarcation potential.Growth temperature is 300 DEG C, grows the laser frequency of middle layer SRO
Rate is 5HZ；Laser energy is 80mj；Partial pressure of oxygen is 15pa；Growth time is 24min.Laser frequency is 5HZ when growing BFO；
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 is cultivated without stem cell is supplemented between Osteoinductive Factor with money
Base, adherent growth, every 3d are digested and are passed on 0.25% pancreatin in the culture bottle of T75 after MSCs cell recovery.
(3) induction MSCs is to Osteoblast Differentiation: take 3-6 to be inoculated in the surface BFO of different polarization directions for MSCs, using without at
The culture medium of bone-inducing factor is placed in 37 DEG C of temperature, 5% CO2It is cultivated in incubator.
(4) MSCs sticks observation, including cell spreading area and focal adhension differential expression after cultivating 3h.
(5) MSCs Osteoblast Differentiation detects after cultivating 1d, the immunofluorescence dyeing including BMP2, RUNX2.
It is 70-80nm by the resulting BFO film thickness of above step, uniformity is poor, and crystal grain arrangement is relatively mixed and disorderly and table
There are bulky grains in face.Intermediate layer thickness is 70-80nm, and STO substrate thickness is 0.5mm, and surface potential is -30~-45mV, induction
MSCs Osteoblast Differentiation index BMP2, RUNX2 expression is low.
Embodiment 3
(1) electroactive material bismuth ferrite (BFO) film of preparation simulation demarcation potential.Using STO as substrate, swashed using pulse
The BFO epitaxial film of Photodeposition (PLD) simulation demarcation potential.Growth temperature is 500 DEG C, grows the laser frequency of middle layer SRO
Rate is 5HZ；Laser energy is 95mj；Partial pressure of oxygen is 20pa；Growth time is 2min.Laser frequency is 5HZ when growing BFO；
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 is cultivated without stem cell is supplemented between Osteoinductive Factor with money
Base, adherent growth, every 3d are digested and are passed on 0.25% pancreatin in the culture bottle of T75 after MSCs cell recovery.
(3) induction MSCs is to Osteoblast Differentiation: take 3-6 to be inoculated in the surface BFO of different polarization directions for MSCs, using without at
The culture medium of bone-inducing factor is placed in 37 DEG C of temperature, 5% CO2It is cultivated in incubator.
(4) MSCs sticks observation, including cell spreading area and focal adhension differential expression after cultivating 3h.
(5) MSCs Osteoblast Differentiation detects after cultivating 1d, the immunofluorescence dyeing including BMP2, RUNX2.
By the resulting simulation demarcation potential BFO film thickness of above step be 5-10nm, uniformity is poor, compactness compared with
It is low.For the LSMO or SRO of middle layer with a thickness of 5-10nm, STO substrate thickness is 0.5mm, and surface potential is -45~-60mV, induction
MSCs Osteoblast Differentiation index BMP2, RUNX2 expression is lower.
Claims (6)
1. a kind of material for promoting mescenchymal stem cell Osteoblast Differentiation, it is characterized in that the material is the ruthenium using strontium titanates as substrate
Sour strontium is middle layer, and surface deposits the film of bismuth ferrite；For the bismuth ferrite thin film with a thickness of 20 ~ 30nm, middle layer ruthenic acid strontium is thick
Degree is 20 ~ 30nm, and surface potential is -65 ~ -70mV.
2. promote the preparation method of the material of mescenchymal stem cell Osteoblast Differentiation as described in claim 1, it is characterized in that using
Pulsed laser deposition.
3. the preparation method of the material according to claim 2 for promoting mescenchymal stem cell Osteoblast Differentiation, it is characterised in that
Growth temperature is 300-800 DEG C in the preparation process, and the laser frequency of growth middle 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.
4. the material of mescenchymal stem cell Osteoblast Differentiation is promoted to promote mescenchymal stem cell skeletonization point as described in claim 1
Application in change, it is characterized in that including the following steps: that (1) preparation promotes the material model of mescenchymal stem cell Osteoblast Differentiation；(2)
Mescenchymal stem cell amplification cultivation: using without stem cell media is supplemented between Osteoinductive Factor with money, mescenchymal stem cell is recovered
After cultivate, pancreatin is digested and is passed on；(3) be inoculated with mescenchymal stem cell on material model: taking mescenchymal stem cell to be inoculated in has
The bismuth ferrite thin film surface for the surface charge that polarizes still is trained using the mescenchymal stem cell culture medium without Osteoinductive Factor
It supports；(4) sticking for mescenchymal stem cell is detected with Osteoblast Differentiation.
5. the material according to claim 4 for promoting mescenchymal stem cell Osteoblast Differentiation is promoting mescenchymal stem cell skeletonization
Application in differentiation, it is characterised in that in the step (2), derived from bone marrow or adipose-derived mescenchymal stem cell recovery after in
Adherent growth in the culture bottle of T75 is digested and is passed on 0.25% pancreatin for every 3 days.
6. the material according to claim 5 for promoting mescenchymal stem cell Osteoblast Differentiation is promoting mescenchymal stem cell skeletonization
Application in differentiation, it is characterised in that in the step (3), 3-6 is taken to be inoculated in bismuth ferrite surface for mescenchymal stem cell, used
Culture medium without Osteoinductive Factor is placed in 37 DEG C of temperature, is cultivated in 5% CO2 incubator.
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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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| CN104099295A (en) * | 2014-07-07 | 2014-10-15 | 暨南大学 | Application of magnetic nano materials in promotion of mesenchymal stem cell osteogenic differentiation |
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