CN112159791A - Method for promoting directional osteogenic differentiation of mesenchymal stem cells - Google Patents
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Abstract
The invention relates to a method for promoting mesenchymal stem cells to differentiate directionally into bone, which solves the technical problem that the existing implant material can not provide in vitro dynamic regulation and control surface potential after implantation to regulate cell function and differentiation, and comprises the following steps: macrophage amplification culture; performing amplification culture on the mesenchymal stem cells; regulating macrophage polarization and osteogenic differentiation of mesenchymal stem cells: inoculating macrophages on the surface of an electrified bionic implantation membrane material with a magnetoelectric coupling effect, and inoculating mesenchymal stem cells on a co-culture chamber; transferring the co-culture chamber inoculated with the mesenchymal stem cells into a charged bionic membrane material culture plate placed with inoculated macrophages to establish an indirect co-culture system; the external magnetic field is changed to cause the change of the magnetoelectric microenvironment and regulate and control the polarization of macrophages, thereby promoting the osteogenic differentiation of the mesenchymal stem cells. The invention can be used for promoting the directional osteogenic differentiation of the mesenchymal stem cells.
Description
Technical Field
The invention relates to a method for promoting cell differentiation, in particular to a method for promoting mesenchymal stem cells to differentiate directionally into bone.
Background
With the recent economic development, the number of cases of bone defects due to factors such as traumatic tumor has increased. Currently, bone defect repair is often performed by implantation. The implant can be divided into autogenous bone, allogenic bone and artificial synthetic material. The autologous bone transplantation often has the problems of insufficient bone mass in a supply area, secondary infection in an operation area and the like. The donor of the allogeneic bone is less, and the risk of immunological rejection exists. In order to solve the problems, artificial synthetic materials are adopted to replace bone tissues for implantation repair, and the method is a research hotspot of the current tissue engineering. Researchers have found that there is a natural electromagnetic microenvironment in the human body and that it plays an important role in the growth and regeneration of tissues. In order to simulate the electromagnetic environment in vivo, the method of repairing the defect of the bone tissue by adopting electromagnetic stimulation is widely concerned. Currently, the common method is to apply an electromagnetic field or implant a charged or magnetic biological material in vivo. However, the conventional methods still have the defects that the stimulation area cannot be accurately positioned, the potential is attenuated after the material is implanted, the in-vitro regulation and control cannot be realized after the material is implanted, and the like.
Bone tissue regeneration is a complex physiological process involving multiple cells, such as macrophages and mesenchymal stem cells. Macrophages, the first cells to reach the defect area, play an indispensable role in the early osteogenesis process. Macrophages can be classified into pro-inflammatory type (M1) and anti-inflammatory type (M2), wherein M2 type macrophages can secrete multiple cytokines to promote bone tissue remodeling. Therefore, inducing macrophage differentiation to M2 type accelerates the process of bone tissue regeneration. The mesenchymal stem cells have the potential of multidirectional differentiation and are seed cells commonly used in tissue engineering. The method adopts proper electromagnetic stimulation to regulate macrophage polarization to M2 type and secrete osteogenesis related cell factor, thereby inducing mesenchymal stem cells to differentiate into osteogenesis, and has a wide application prospect in the application of bone tissue regeneration.
The Chinese invention patent application with the patent application number of 201811235132.7 discloses a charged bionic implantation membrane material regulated and controlled by magnetoelectric coupling and a preparation method thereof, wherein the charged bionic implantation membrane material is a film-shaped material prepared from a ferroelectric high-molecular polymer, a magnetic particle filler and an organic solvent, and solves the technical problems of potential attenuation and unstable long-term repair effect of the existing charged biological material after long-term implantation, but the implantation material can not provide the in-vitro dynamic regulation and control of surface potential after implantation to regulate cell function and differentiation.
Disclosure of Invention
The invention provides an electromagnetic material which can be regulated and controlled after implantation, aiming at the technical problem that the existing implantation material can not provide the in-vitro dynamic regulation and control surface potential after implantation so as to regulate the cell function and differentiation, thereby inducing macrophage M2 polarization and promoting the directional osteogenic differentiation of mesenchymal cells.
Therefore, the invention provides a method for promoting the directional osteogenic differentiation of mesenchymal stem cells, which comprises the following steps: (1) macrophage amplification culture; (2) performing amplification culture on the mesenchymal stem cells; (3) regulating macrophage polarization and osteogenic differentiation of mesenchymal stem cells: inoculating macrophages on the surface of an electrified bionic implantation membrane material with a magnetoelectric coupling effect, and inoculating mesenchymal stem cells on a co-culture chamber; (4) transferring the co-culture chamber inoculated with the mesenchymal stem cells into a charged bionic membrane material culture plate placed with inoculated macrophages to establish an indirect co-culture system; (5) the external magnetic field is changed to cause the change of the magnetoelectric microenvironment and regulate and control the polarization of macrophages, thereby promoting the osteogenic differentiation of the mesenchymal stem cells.
Preferably, in the step (1), the macrophage is macrophage Raw 264.7.
Preferably, in the step (1), the macrophage Raw264.7 amplification culture adopts a high-glucose DMEM culture medium without induction factors, the macrophage Raw is subjected to adherent culture in a T25 culture flask, the cells are cultured after recovery, and the cells are subjected to subculture by adding the culture medium and adopting a blowing beating mode.
Preferably, in the step (2), the mesenchymal stem cell expansion culture: and (3) adopting an alpha-MEM culture medium without induction factors, culturing MSCs after recovery, and carrying out passage in a pancreatin digestion mode with the mass-volume ratio of 0.25% for 2-3 days.
Preferably, in the step (4), 8-15 generations of macrophages are inoculated on the charged biomimetic membrane material, and 3-5 generations of mesenchymal stem cells are inoculated on the co-culture(transwell) in the chamber, an indirect CO-culture system was constructed using medium without induction factor, placed at 37 ℃ in a humidity of 95% and 5% by volume of CO2And culturing in an incubator.
Preferably, in the step (5), the magnetoelectric microenvironment of the cells is changed by changing the applied magnetic field every 12 hours.
The invention has the following beneficial effects:
the invention dynamically regulates and controls CoFe through an external magnetic field2O4The surface potential of a/P (VDF-TrFE) material provides a dynamic magnetoelectric microenvironment to induce macrophage M2 type differentiation and mesenchymal stem cell directed differentiation, and the problems that the existing electromagnetic implantation material cannot realize dynamic regulation and induce cell polarization and directed differentiation are solved; CoFe used in the invention2O4the/P (VDF-TrFE) material has good magnetoelectric coupling performance, and the surface potential of the material can be regulated and controlled through external magnetic fields with different sizes, so that the optimal electromagnetic stimulation environment is provided for the directional differentiation and polarization of cells; the invention achieves the aims of inducing macrophage M2 polarization and mesenchymal cell oriented osteogenic differentiation without any biological and chemical inducing factors, and has better safety and controllability.
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FIG. 1 is CoFe implanted in vivo as described in example 12O4Scanning electron microscopy of/P (VDF-TrFE) material.
FIG. 2 is CoFe implanted in vivo as described in example 12O4Surface potential diagrams induced under different applied magnetic field conditions for/P (VDF-TrFE) materials.
FIG. 3A depicts CoFe implanted in vivo as described in example 12O4A macrophage M2 type differentiation surface marker flow detection result chart under the dynamic regulation and control of a VDF-TrFE material and an external magnetic field.
FIG. 3B is a graph showing the results of flow measurement of the differentiation surface marker of macrophage M2 type in the control group described in example 1.
FIG. 4A depicts CoFe implanted in vivo as described in example 12O4Dynamic control of/P (VDF-TrFE) material and external magnetic fieldNext, the immunofluorescence detection result chart of macrophage M2 type differentiation surface marker.
FIG. 4B is a graph showing the immunofluorescence assay results for macrophage M2-type differentiation surface marker in the control group described in example 1.
FIG. 5A depicts CoFe implanted in vivo as described in example 12O4And a mesenchymal stem cell osteogenic differentiation marker immunofluorescence detection result diagram under dynamic regulation and control of a/P (VDF-TrFE) material and an external magnetic field.
Fig. 5B is a graph showing immunofluorescence detection results of osteogenic differentiation markers of mesenchymal stem cells in the control group according to example 1.
FIG. 6A depicts CoFe implanted in vivo as described in example 12O4And a mesenchymal stem cell calcium nodule alizarin red staining result graph under dynamic regulation and control of a/P (VDF-TrFE) material and an external magnetic field.
Fig. 6B is a graph showing the result of alizarin red staining on calcium nodule of mesenchymal stem cell in the control group described in example 1.
Detailed Description
The present invention will be further described with reference to the following examples.
However, the above description is only exemplary of the present invention, and the scope of the present invention should not be limited thereby, and the replacement of the equivalent components or the equivalent changes and modifications made according to the protection scope of the present invention should be covered by the claims of the present invention.
The charged biomimetic membrane capable of realizing dynamic regulation and control of magnetoelectric microenvironment used by the invention is CoFe2O4The preparation method of the/P (VDF-TrFE) nano-membrane is disclosed in the Chinese patent application with the patent application number of 201811235132.7 and the name of the charged bionic implantation membrane material regulated and controlled by magnetoelectric coupling and the preparation method thereof.
Tests prove that the material is in CoFe2O4When the proportion of the filler is 10 wt.%, the filler has the best magnetoelectric coupling performance, and the magnetoelectric coupling coefficient can reach 6.08mV-1.Oe-1. Can be converted according to the magnetoelectric coupling coefficient, and can regulate and control CoFe when the external magnetic field is 2300Oe2O4The proportion of filler being 10 wt.%The surface potential of the material is 54 mV. According to experiments, 54mV of surface potential is the optimal induced osteogenesis potential. Therefore, the invention uses an external magnetic field with the size of 2300Oe to perform dynamic magnetoelectricity microenvironment stimulation.
After the cells are inoculated and placed in a co-culture system, the cells are stimulated by a dynamic electromagnetic microenvironment which is changed every 12 hours. After culturing for 24 hours, detecting the M2 type macrophage surface marker CD206 by flow cytometry, wherein the positive rate of a group providing a dynamic magnetoelectric microenvironment is 56 percent, and the positive rate of a control group (without materials and external magnetic fields) is 17.8 percent (the flow experiment control group is shown in figure 3B); detecting the M2 type macrophage surface marker CD206 by immunofluorescence staining, wherein the fluorescence intensity of a group providing a dynamic magnetoelectric microenvironment is remarkably higher than that of a control group (the control group of a CD206 immunofluorescence experiment is shown in figure 4B); after culturing for 7 days, detecting the expression condition of osteogenic differentiation related protein RUNX2 through immunofluorescence, wherein the fluorescence intensity of the mesenchymal stem cells providing a dynamic magnetoelectric microenvironment is obviously higher than that of a control group (the RUNX2 immunofluorescence experiment control group is 5B); after 21 days of culture, alizarin red staining was performed, and mature calcium nodules were observed in the group providing the dynamic magnetoelectric microenvironment, whereas the control group did not (alizarin red experimental control group is fig. 6B).
Example 1
(1) Amplification culture of mouse macrophage (macrophage Raw264.7): adopting a high-glucose DMEM culture medium without induction factors, recovering Raw264.7 cells, carrying out adherent culture in a T25 culture bottle, flushing with PBS every 1-2 days, replacing with a new culture medium (the high-glucose DMEM culture medium), and gently blowing and beating the adherent cells for passage.
(2) And (3) mesenchymal stem cell amplification culture: recovering Mesenchymal Stem Cells (MSCs) by using an alpha-MEM culture medium without induction factors, carrying out adherent culture in a T25 culture bottle, and carrying out passage by using a 0.25% trypsin digestion mode every 2-3 days.
(3) Inducing macrophage M2 type polarization and mesenchymal stem cell oriented osteogenic differentiation: inoculating 8-15 generation macrophage Raw264.7 on charged bionic membrane material, inoculating 3-5 generation MSCs in co-culture (transwell) chamber (Millipore, USA) to construct indirect co-culture system, adopting culture medium without inducing factor,placing at 37 deg.C, humidity of 95%, and CO volume fraction of 5%2And culturing in an incubator. After the cells are attached to the wall, the cells are subjected to dynamic electromagnetic microenvironment stimulation (dynamic regulation and control through an external magnetic field) changed every 12 hours.
(4) Detecting macrophage M2 type polarization condition: after further incubation for 24 hours, the macrophage surface marker CD206 of M2 type was detected by flow cytometry, and cell morphology and macrophage surface marker CD206 were observed by immunofluorescence. Detecting the osteogenic differentiation condition of the MSCs: after the culture is continued for 7 days, the expression condition of the osteoblastic differentiation related protein RUNX2 of the MSCs is detected by immunofluorescence. After 21 days of culture, alizarin red staining was performed and calcium nodule formation was observed.
(5) In the macrophages obtained by the steps, the positive rate of the M2 type macrophage surface marker CD206 is 56%, and the positive rate of the M2 type macrophage surface marker CD206 in a control group (without materials and external magnetic fields) is only 17.8%;
among the macrophages obtained by the steps, the fluorescence intensity of an experimental group M2 type macrophage surface marker CD206 stimulated by a dynamic electromagnetic microenvironment is stronger, and the fluorescence intensity of a control group CD206 is obviously weaker than that of the experimental group;
in the MSCs obtained by the steps, the fluorescence intensity of the bone formation related protein RUNX2 of the MSCs in the experimental group stimulated by the dynamic electromagnetic microenvironment is obviously stronger, and the fluorescence intensity of the RUNX2 in the control group is obviously weaker than that of the experimental group;
the MSCs obtained by the above steps were subjected to alizarin red staining, and mature calcium nodule formation was observed in the experimental group using dynamic electromagnetic microenvironment stimulation, compared to the binned group, and no mature calcium nodule formation was observed in the control group.
Claims (6)
1. A method for promoting mesenchymal stem cells to differentiate directionally into bone is characterized by comprising the following steps:
(1) macrophage amplification culture;
(2) performing amplification culture on the mesenchymal stem cells;
(3) regulating macrophage polarization and osteogenic differentiation of mesenchymal stem cells: inoculating macrophages on the surface of an electrified bionic implantation membrane material with a magnetoelectric coupling effect, and inoculating mesenchymal stem cells on a co-culture chamber;
(4) transferring the co-culture chamber inoculated with the mesenchymal stem cells into a charged bionic membrane material culture plate placed with inoculated macrophages to establish an indirect co-culture system;
(5) the external magnetic field is changed to cause the change of the magnetoelectric microenvironment and regulate and control the polarization of macrophages, thereby promoting the osteogenic differentiation of the mesenchymal stem cells.
2. A method for promoting mesenchymal stem cell directed osteogenic differentiation according to claim 1, wherein in the step (1), the macrophage is macrophage Raw 264.7.
3. The method for promoting mesenchymal stem cell directed osteogenic differentiation according to claim 2, wherein in the step (1), the macrophage Raw264.7 amplification culture adopts high-sugar DMEM medium without induction factor, the macrophage Raw is subjected to adherent culture in a T25 culture flask, the cells are cultured after recovery, and the cells are subjected to passage by adding the culture medium in a blowing and beating manner.
4. A method for promoting directional osteogenic differentiation of mesenchymal stem cells according to claim 1, wherein in the step (2), the mesenchymal stem cells are cultured in an amplification way: and (3) promoting the recovery of mesenchymal stem cells by adopting an alpha-MEM culture medium without an induction factor, culturing, and carrying out passage by means of trypsinization.
5. The method for promoting directional osteogenic differentiation of mesenchymal stem cells according to claim 1, wherein in the step (4), 8-15 generations of macrophages are inoculated on the charged biomimetic membrane material, 3-5 generations of mesenchymal stem cells are inoculated in the co-culture chamber, an indirect co-culture system is constructed, and a culture medium without induction factors is adopted and placed in an incubator for culture.
6. The method for promoting osteogenic differentiation of mesenchymal stem cells according to claim 1, wherein in the step (5), the magnetoelectric microenvironment of the cells is changed by changing the applied magnetic field every 12 hours.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113430169A (en) * | 2021-07-01 | 2021-09-24 | 北京大学口腔医学院 | Method for regulating macrophage differentiation |
| CN113604544A (en) * | 2021-08-03 | 2021-11-05 | 北京大学口腔医学院 | Biological material function prediction evaluation method |
| CN114891728A (en) * | 2022-04-07 | 2022-08-12 | 广东医科大学附属医院 | Polyelectrolyte membrane, macrophage exosome and application of polyelectrolyte membrane and macrophage exosome in promotion of BMSCs differentiation |
| 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 |
| CN115232785A (en) * | 2022-09-21 | 2022-10-25 | 北京大学口腔医学院 | Compositions, methods and bone repair uses for promoting osteogenic differentiation of mesenchymal stem cells |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113430169A (en) * | 2021-07-01 | 2021-09-24 | 北京大学口腔医学院 | Method for regulating macrophage differentiation |
| CN113604544A (en) * | 2021-08-03 | 2021-11-05 | 北京大学口腔医学院 | Biological material function prediction evaluation method |
| CN113604544B (en) * | 2021-08-03 | 2023-03-10 | 北京大学口腔医学院 | Biological material function prediction and evaluation method |
| 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 |
| CN114891728A (en) * | 2022-04-07 | 2022-08-12 | 广东医科大学附属医院 | Polyelectrolyte membrane, macrophage exosome and application of polyelectrolyte membrane and macrophage exosome in promotion of BMSCs differentiation |
| CN114891728B (en) * | 2022-04-07 | 2023-01-03 | 广东医科大学附属医院 | Polyelectrolyte membrane, macrophage exosome and application of polyelectrolyte membrane and macrophage exosome in promotion of BMSCs differentiation |
| CN115232785A (en) * | 2022-09-21 | 2022-10-25 | 北京大学口腔医学院 | Compositions, methods and bone repair uses for promoting osteogenic differentiation of mesenchymal stem cells |
| CN115232785B (en) * | 2022-09-21 | 2022-12-13 | 北京大学口腔医学院 | Composition, method and bone repair use for promoting osteogenic differentiation of mesenchymal stem cells |
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