CN110819588A - Use of piezoelectric materials for stem cell proliferation and/or differentiation - Google Patents
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Abstract
The invention discloses application of a piezoelectric material in proliferation and/or differentiation of stem cells, wherein the piezoelectric effect of the piezoelectric material is excited by ultrasonic waves. When the piezoelectric material is applied to proliferation and/or differentiation of stem cells, the activity and dryness of the stem cells are not reduced, and meanwhile, good biocompatibility can be kept to promote the proliferation of the stem cells; moreover, by adopting the ultrasonic wave to excite the piezoelectric effect of the piezoelectric material, the mechanical signal of the ultrasonic wave can be converted into an electric signal to be provided for the stem cells, which is beneficial to inducing the directional differentiation of the stem cells.
Description
Technical Field
Embodiments of the present invention relate to the field of biomaterials, in particular to the use of piezoelectric materials for stem cell proliferation and/or differentiation.
Background
Proliferation and differentiation of stem cells are central and major targets in the field of tissue engineering and regeneration. Studies have shown that biomaterial-mediated physical factors (e.g., material hardness, surface morphology, and surface charge) and chemical factors (chemical composition) can influence the fate of stem cell differentiation. For example, the neural cells differentiated from the stem cells are electrically active cells, and neural differentiation of the stem cells can be performed based on the surface charge. The piezoelectric material has good biocompatibility and piezoelectric effect of generating electric charge by stress, and has potential application prospect in the research field of proliferation and differentiation of stem cells.
However, the piezoelectric material has uncontrollable mechanical force movement in the living body, which greatly limits the practical application of the piezoelectric material in tissue engineering, and has defects in regulating stem cell proliferation and differentiation.
Disclosure of Invention
In view of the above technical problems, embodiments of the present invention propose the use of piezoelectric materials in stem cell proliferation and/or differentiation under the synergistic effect of ultrasound.
According to one aspect of the present invention, the use of a piezoelectric material for the proliferation and/or differentiation of stem cells is proposed, wherein the piezoelectric effect of the piezoelectric material is excited using ultrasound.
According to some embodiments, the stem cell is differentiated into a neural cell.
According to some embodiments, the stem cell is a neural stem cell and/or a mesenchymal stem cell.
According to some embodiments, the piezoelectric material enters a stem cell, and the ultrasound acts on the stem cell that has ingested the piezoelectric material.
According to some embodiments, the stem cells are exposed to the piezoelectric material for 12-24 hours before the application of ultrasound, and preferably, the stem cells are exposed to the piezoelectric material for 24 hours before the application of ultrasound.
According to some embodiments, the frequency of the ultrasound is 1MHz or 3 MHz; the power density of the ultrasonic wave is 0.1-3W/cm2Preferably 0.2 to 1W/cm2(ii) a The service time of the ultrasonic wave is 5-150s, preferably 10 s.
According to some embodiments, the ultrasound is used 1-3 times a day, preferably 1 time a day.
According to some embodiments, the ultrasound is used at a temperature of 20-37 ℃.
According to some embodiments, the piezoelectric material comprises one of tetragonal barium titanate, zinc oxide, tetragonal lithium niobate, boron nitride; and the morphology of the piezoelectric material is nano-particles and/or nano-rods.
According to some embodiments, the nanoparticles of the piezoelectric material have a particle size of 10-500 nm.
According to some embodiments, the piezoelectric material is dispersed in a phosphate buffer, the concentration of the piezoelectric material being 10-200 μ g/mL, preferably 50 μ g/mL. .
In the application of the piezoelectric material in stem cell proliferation and/or differentiation according to the embodiment of the invention, when the piezoelectric material is applied to stem cell proliferation and/or differentiation, the activity and the dryness of stem cells are not reduced, and meanwhile, good biocompatibility can be maintained to promote the proliferation of the stem cells; moreover, by adopting the ultrasonic wave to excite the piezoelectric effect of the piezoelectric material, the mechanical signal of the ultrasonic wave can be converted into an electric signal to be provided for stem cells, thereby being beneficial to inducing the directional differentiation of the cells.
Drawings
Other objects and advantages of the present invention will become apparent from the following description of the invention which refers to the accompanying drawings, and may assist in a comprehensive understanding of the invention.
Fig. 1 shows a scanning electron micrograph of barium titanate nanoparticles according to an exemplary embodiment of the present invention;
FIG. 2 shows a transmission electron micrograph of the barium titanate nanoparticles of FIG. 1;
FIG. 3 shows an X-ray diffraction pattern of the barium titanate nanoparticles of FIG. 1;
FIG. 4 shows the effect of barium titanate on the proliferation status of stem cells;
FIG. 5 shows bright field photographs of stem cells after barium titanate addition;
FIG. 6 shows immunofluorescence staining results after neural differentiation of stem cells; and
FIG. 7 is a schematic diagram showing the results of expression of marker genes after neural differentiation of stem cells.
Detailed Description
The technical scheme of the invention is further specifically described by the following embodiments and the accompanying drawings. In the following detailed description, for purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the embodiments of the disclosure. It may be evident, however, that one or more embodiments may be practiced without these specific details.
The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.
According to one aspect of the present invention, the use of a piezoelectric material for the proliferation and/or differentiation of stem cells is proposed, wherein the piezoelectric effect of the piezoelectric material is excited using ultrasound. In the application of the piezoelectric material in stem cell proliferation and/or differentiation according to the embodiment of the invention, when the piezoelectric material is applied to stem cell proliferation and/or differentiation, the activity and the dryness of stem cells are not reduced, and meanwhile, good biocompatibility can be maintained to promote the proliferation of the stem cells; moreover, by adopting the ultrasonic wave to excite the piezoelectric effect of the piezoelectric material, the mechanical signal of the ultrasonic wave can be converted into an electric signal to be provided for the cells, which is beneficial to inducing the directional differentiation of the stem cells. Therefore, the piezoelectric material has a wide practical prospect in proliferation and/or differentiation of stem cells under the action of ultrasonic waves as a biological material with a piezoelectric effect.
Stem cells are primitive and unspecified cells that have the ability to self-replicate on the one hand and differentiate into a variety of functional cells on the other hand, with the potential function of regenerating various tissues and organs. The stem cells of the embodiments of the invention may be various types of stem cells, including, for example, but not limited to, neural stem cells and/or mesenchymal stem cells. Neural stem cells refer to a cell population that exists in the nervous system, has the potential to differentiate into neurons, astrocytes and oligodendrocytes, thereby being capable of producing a large amount of brain cells and performing self-renewal, and sufficiently providing a large amount of brain cells. Mesenchymal stem cells can be differentiated into various tissue cells such as fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, cardiac muscle, endothelium and the like under specific induction conditions in vivo or in vitro. The 'tissue engineering' of constructing tissues or organs in vitro or in vivo based on stem cells provides a perfect solution for the repair of damaged tissues and organs of human bodies. For example, neural differentiation of stem cells (i.e., differentiation into neural cells) can be used to repair neural injury. Further, since neural cells are electrically active cells whose functions are closely related to the surface charge conditions, neural differentiation of stem cells can be promoted based on control of the electrical activity of the cells.
Piezoelectric materials are a class of crystalline materials with non-centrosymmetric structures, with electromechanical coupling properties. When the material is acted by mechanical force, the material is deformed, the positive and negative charge centers in the material shift, and charges with opposite signs are generated at two ends of the material, namely, the piezoelectric effect. The piezoelectric effect of the piezoelectric material is utilized to adjust the electrical properties of the stem cell by applying a force to the piezoelectric material to generate an electrical charge. In addition, the piezoelectric material has good biocompatibility, and the activity and the dryness of stem cells are not reduced. Furthermore, in order to better control the stress condition of the piezoelectric material, the piezoelectric effect of the piezoelectric material can be excited by ultrasonic waves. Specifically, the piezoelectric material may convert a mechanical signal of the ultrasonic wave into an electrical signal to be provided to the stem cell to induce neural differentiation of the stem cell. Therefore, mechanical signals obtained by the piezoelectric material can be effectively regulated and controlled by regulating various indexes of the ultrasonic waves, and the electrical property of the stem cells is further regulated to promote directional differentiation.
When the piezoelectric material is applied to proliferation and/or differentiation of stem cells, the stem cells can phagocytose the piezoelectric material into the cells due to the good biocompatibility of the piezoelectric material, and then the ultrasonic waves can act on the stem cells which take in the piezoelectric material. In the embodiment of the present invention, the piezoelectric material may be added 12 hours after stem cells are attached, and the piezoelectric material is generally dispersed in a phosphate buffer, and the concentration of the piezoelectric material is not particularly limited, and may be, for example, 10 to 200 μ g/mL, and further may preferably be 50 μ g/mL. Generally, the stem cells are exposed to the piezoelectric material for 12 to 24 hours and then the ultrasonic waves are applied, and preferably, the stem cells are exposed to the piezoelectric material for 24 hours and then the ultrasonic waves are applied.
Further, the piezoelectric material may include one of tetragonal barium titanate, zinc oxide, tetragonal lithium niobate, boron nitride, and the morphology of the piezoelectric material may be nanoparticles and/or nanorods, and the nanoparticles may be nanocubes or round grains or any suitable morphology. The size of the particle size of the nanoparticles is not limited, and preferably, the particle size may range from 10 to 500 nm.
In particular, barium titanate material has a high dielectric constant and low dielectric loss, is one of the most widely used materials in electronic ceramics, and is known as "pillar of electronic ceramics industry". Most of the current research on barium titanate materials focuses on electronic devices, and the research in the field of biology is limited to the biocompatibility of different cell lines and the osteogenic differentiation level of stem cells, and the research in the neural differentiation direction of the stem cells is lacked. In the embodiment of the invention, the barium titanate material has good biocompatibility with the stem cell, can be swallowed by the stem cell, and can induce the neural differentiation of the stem cell under the action of ultrasonic waves.
According to an exemplary embodiment of the present invention, barium titanate nanoparticles may be prepared as follows:
s11, firstly weighing 392mg of nano titanium dioxide powder and 75.6g of barium hydroxide octahydrate in a reaction kettle, then pouring 20mL of deionized water, and reacting for 5 days at 200 ℃;
s12, taking out the reaction kettle after the reaction while the reaction kettle is hot, pouring out the alkali liquor while the alkali liquor is liquid, and leaving a small amount of alkali liquor and barium titanate at the bottom of the reaction kettle;
s13, repeatedly washing barium titanate with distilled water, neutralizing residual barium hydroxide with hydrochloric acid, and adjusting the pH value of the barium titanate aqueous solution to 6.5-7.0;
s14, carrying out suction filtration on the barium titanate aqueous solution with the adjusted PH value, and repeatedly cleaning the barium titanate again in the suction filtration process;
s15, placing the filtered barium titanate and the filter membrane together in a 60 ℃ drying oven for drying;
and S16, grinding and dispersing the dried barium titanate in an agate mortar, weighing 50 mu g of the barium titanate, putting the barium titanate into a 1.5mL centrifuge tube, adding 1mL of ethanol solution with the volume fraction of 75%, and performing ultrasonic dispersion.
Fig. 1 shows a scanning electron microscope image of the barium titanate nanoparticles prepared as above, from which it can be seen that the particle size of the barium titanate nanoparticles is 80-160nm, and the barium titanate nanoparticles prepared as above by the hydrothermal synthesis method are tetragonal or irregular spheres. FIG. 2 shows a transmission electron micrograph of the barium titanate nanoparticles of FIG. 1, the barium titanate nanoparticles having interplanar spacings of 0.398nm and 0.402nm, corresponding to the crystallographic planes of tetragonal barium titanate, observed by high resolution transmission electron microscopy (commercially available from JEOL, Japan model JEM 2100). Further, the barium titanate powder was subjected to X-ray diffraction analysis, and the diffraction pattern was as shown in fig. 3, and by analyzing the diffraction peaks, it was confirmed that the synthesized barium titanate was tetragonal crystal, and splitting of the (200) diffraction peak was observed on the diffraction pattern.
According to another exemplary embodiment of the present invention, zinc oxide nanorods may be prepared as follows:
s21, dissolving 0.0439g of zinc acetate dihydrate in 20mL of anhydrous methanol, and stirring for 10min at 60 ℃ by using a magnetic stirrer;
s22, dissolving 0.0336g of potassium hydroxide in 20mL of anhydrous methanol, and magnetically stirring at room temperature for 10 min;
s23, dropwise adding the solution in the step S22 into the solution in the step S21, and stirring the mixed solution strongly at 60 ℃ for 2 hours to ensure that the reaction is sufficient;
s24, soaking the substrate for growing the zinc oxide in the mixed solution for 100S;
s25, adding a hexamethylenetetramine aqueous solution (32mmol/L) and zinc nitrate hexahydrate (32mmol/L) into a reaction kettle, and reacting for 12 hours at 100 ℃;
and S26, washing with deionized water after the reaction is finished, and drying at 60 ℃.
Further, taking barium titanate as an example, the invention detects the biocompatibility of the piezoelectric material.
The control group was specified as follows: the stem cells were treated at 1.3 x 104The density of each hole is inoculated in a confocal culture dish; cells were plated every three days.
Details of the experimental group to which barium titanate was addedComprises the following steps: the stem cells were treated at 1.3 x 104The density of each hole is inoculated in a confocal culture dish; adding barium titanate nano-particle-phosphate buffer solution with the concentration of 50 mug/mL 12 hours after the cells are attached to the wall; cells were plated every three days.
The stem cells of the control group and the experimental group were subjected to cell viability assay using a cell counting kit (CCK-8, Dojindo laboratories), and the results are shown in FIG. 4. As can be seen from fig. 4, the relative proliferation rates of the stem cells of the control group and the experimental group added with barium titanate were not greatly different at days 2, 4 and 6 of the cell culture, i.e., the addition of barium titanate did not affect the cell viability of the stem cells, indicating that the barium titanate nanoparticles have good biocompatibility.
Fig. 5 shows a bright field photograph of the stem cell after barium titanate is added, as shown in fig. 5, barium titanate nanoparticles are engulfed by the stem cell, the amount of barium titanate around the cell is significantly reduced compared to other areas, and a large amount of barium titanate accumulates near the nucleus and in the cytoskeleton.
Further, the ultrasonic wave for exciting the piezoelectric effect of the piezoelectric material according to the present invention may be generated by an ultrasonic transfectant apparatus, and in addition, other apparatuses capable of generating the ultrasonic wave include a B-ultrasonic machine, an ultrasonic therapeutic apparatus, and the like. Relevant indexes of the ultrasonic waves can influence stem cell adhesion and endocytosis piezoelectric materials. In an embodiment of the present invention, the frequency of the ultrasonic wave may be 1MHz or 3 MHz; the power density of the ultrasonic wave can be 0.1-3W/cm2Preferably 0.2 to 1W/cm2(ii) a The service time of the ultrasonic wave can be 5-150s, preferably 10 s; the frequency of use of the ultrasonic wave may be 1 to 3 times a day, preferably 1 time a day; and the use temperature of the ultrasonic wave can be 20-37 ℃.
The following description is based on specific examples.
Example 1
The barium titanate nanoparticles were prepared using the above method of preparing the barium titanate nanoparticles of fig. 1-3, followed by the following steps:
step one, 1.3 x 10 of stem cells are added4The density of each hole is inoculated in a confocal culture dish;
step two, adding barium titanate nano-particle-phosphate buffer solution with the concentration of 50 mug/mL after stem cells adhere to the wall for 12 hours;
step three, adding barium titanate for 12 hours, and then applying 0.8W/cm to stem cells210s of ultrasonic waves;
and step four, repeating the step three, wherein the solution is changed every three days for the cells.
Examples 2-3 similar procedure as in example 1 was followed, except that:
example 2 adding barium titanate in step three for 12 hours and then applying 1.2W/cm to the stem cells210s of ultrasonic waves;
example 3 addition of barium titanate in step three for 12 hours followed by application of 0.8W/cm to the cells2The ultrasonic wave of (5) for 60 s.
The stem cells of examples 1-3 were characterized and microscopically observed that barium titanate nanoparticles were engulfed by the cells, the barium titanate surrounding the cells was significantly reduced in number compared to other areas, and a large amount of barium titanate was accumulated near the nucleus and in the cytoskeleton. In addition, it was microscopically observed that the cell morphology was disrupted and the number of apoptosis was increased in example 2; the cell morphology was clearly disrupted in example 3 and the number of apoptotic cells was significantly increased. It should be noted that, in practical application, the ultrasonic waves penetrate the skin or other tissues of the human body and act on the stem cells with a barrier, which is different from the embodiment 1-3 in which the ultrasonic waves act on the cells directly from the outside of the cell membrane, so that the intensity of the ultrasonic waves actually acting on the stem cells in clinical application is reduced without damaging the cells. Therefore, in example 2, 1.2W/cm2The power density of the ultrasonic wave and the use time of the ultrasonic wave of 60s in the embodiment 3 still fall into the protection scope of the claims of the present invention.
Further, after the stem cells of example 1 were cultured for two weeks, the cells were subjected to immunofluorescence staining of neuronal and glial cells. The method comprises the following specific steps:
soaking the stem cells in paraformaldehyde with a volume fraction of 4% for 15 minutes to fix the cells, and then washing the cells with PBS buffer for 3 times, 5 minutes each time;
then treating the cells with 0.1% triton solution by volume fraction for 5 minutes, and washing with buffer solution for three times, five minutes each time;
then, sealing nonspecific sites of the cells by goat serum with the volume fraction of 1%, and sealing for 1.5 hours at room temperature;
diluting the anti-Tuj1 antibody of murine origin (Abcam Corp.)/anti-GFAP antibody of rabbit origin (Abcam Corp.)/anti-MAP-2 antibody of rabbit origin (Abcam Corp.) 1000 times, 2000 times and 1000 times respectively in BSA (bovine serum albumin), incubating the cells overnight, and washing with PBS three times for 5 minutes each;
cells were incubated overnight with FITC fluorescence-modified goat/rabbit secondary antibody (Beijing assist holy) overnight, diluted 200-fold with 594-stimulated phalloidin in PBS at room temperature for 4 hours, washed three times with PBS, 5 minutes each;
finally, the cell nuclei were counterstained with DAPI, washed clean with PBS and observed under a fluorescent microscope.
The neuron was labeled with a neuron marker Tuj1, the glial cell was labeled with a glial cell marker GFAP and a microtubule-associated protein MAP-2, and the staining results are shown in fig. 6, in which the first, second, and third rows from top to bottom represent the expression results of Tuj1 protein, GFAP protein, MAP-2 protein, respectively; the first, second, third and fourth columns from left to right represent the superimposed staining results, the staining results of nuclei, cytoskeleton and secondary antibody, respectively. As can be seen from FIG. 6, in example 1, after two weeks of barium titanate synergistic ultrasonication, the expression level of Tuj1 protein in stem cells is significantly higher than that of GFAP protein, and microtubule-associated protein MAP-2 is also expressed. The barium titanate is cooperated with the ultrasonic wave to promote the stem cells to differentiate towards the neuron and the glia.
Example 4
To demonstrate the universality of the piezoelectric material in synergy with ultrasound to promote proliferation and differentiation of stem cells, lithium niobate has also been used in synergy with ultrasound to induce differentiation of stem cells. In this example, genes related to nerves in cells were detected after adding lithium niobate and stimulating for 7 days under the synergistic effect of ultrasonic waves. FIG. 7 shows the expression results of the neuronal marker gene Tuj1, glial marker gene GFAP and microtubule-associated protein MAP-2 after 7 days of cell differentiation, in which the control group was stem cells cultured in the same environment without the addition of piezoelectric material or the application of ultrasound. As can be seen from fig. 7, Tuj1, GFAP and MAP-2 were expressed more sufficiently in the stem cells of this example compared to the control group, indicating that the cells differentiated significantly into the neural cells after the cells had engulfed the lithium niobate nanoparticles and applied the ultrasound for 7 days.
Although the present invention has been described in connection with the accompanying drawings, the embodiments disclosed in the drawings are intended to be illustrative of embodiments of the invention and should not be construed as limiting the invention. Although a few embodiments of the present general inventive concept have been shown and described, it would be appreciated by those skilled in the art that changes may be made in these embodiments without departing from the principles and spirit of the general inventive concept, the scope of which is defined in the claims and their equivalents.
Claims (11)
1. Use of a piezoelectric material for the proliferation and/or differentiation of stem cells, wherein the piezoelectric effect of the piezoelectric material is excited using ultrasound.
2. The use according to claim 1, wherein said stem cells differentiate into neural cells.
3. The use according to any one of claims 1-2, wherein the stem cells are neural stem cells and/or mesenchymal stem cells.
4. Use according to any one of claims 1 to 3, wherein the piezoelectric material is incorporated into stem cells and the ultrasound acts on the stem cells which have taken up the piezoelectric material.
5. Use according to any of claims 1 to 4, wherein the stem cells are exposed to the piezoelectric material for 12 to 24 hours before the application of ultrasound, preferably wherein the stem cells are exposed to the piezoelectric material for 24 hours before the application of ultrasound.
6. Use according to any one of claims 1 to 5, characterized in that the frequency of said ultrasound waves is 1MHz or 3 MHz;
the power density of the ultrasonic wave is 0.1-3W/cm2Preferably 0.2 to 1W/cm2；
The service time of the ultrasonic wave is 5-150s, preferably 10 s.
7. Use according to any of claims 1-6, wherein the ultrasound is used 1-3 times a day, preferably 1 time a day.
8. Use according to any one of claims 1 to 7, wherein the ultrasound is used at a temperature of 20 to 37 ℃.
9. The use according to any one of claims 1-8, wherein the piezoelectric material comprises one of barium tetratitanate, zinc oxide, lithium tetraniobate, boron nitride; and is
The appearance of the piezoelectric material is nano particles and/or nano rods.
10. Use according to claim 9, wherein the nanoparticles of the piezoelectric material have a particle size of 10-500 nm.
11. Use according to any one of claims 1 to 10, wherein the piezoelectric material is dispersed in a phosphate buffer at a concentration of 10 to 200 μ g/mL, preferably 50 μ g/mL.
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