CN115636400B - Preparation method of one-dimensional multifunctional hydroxyapatite nano-belt with secondary structure and application of nano-belt in assembling functional stem cell spheres - Google Patents
Preparation method of one-dimensional multifunctional hydroxyapatite nano-belt with secondary structure and application of nano-belt in assembling functional stem cell spheres Download PDFInfo
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Abstract
The invention relates to a preparation method of a one-dimensional multifunctional hydroxyapatite nano belt with a secondary structure and application thereof on assembling functional stem cell spheres, the method prepares the one-dimensional multifunctional hydroxyapatite nano belt with the secondary structure by a simple method, and the multifunctional hydroxyapatite nano belt is assembled into various types of functional stem cell spheres by culturing and endocytosis of the stem cell spheres, the obtained hydroxyapatite nano belt has a multi-layer stack shape and is bifurcated on the end surface, so that nutrition transportation to the inside of the stem cell spheres is facilitated, the end surface is easy to bifurcation, cell endocytosis is facilitated, cell-material interaction is promoted, the multifunctional stem cell spheres are assembled, the multifunctional stem cell spheres have a faster nerve differentiation speed and a more obvious neuron direction differentiation trend, more mature neurons can be generated in a shorter time, and the nerve tissue repair efficiency is improved to the maximum extent; has the advantages of rapid osteogenic differentiation speed and tissue regeneration promotion.
Description
Technical Field
The invention relates to a preparation method of a one-dimensional multifunctional hydroxyapatite nano belt with a secondary structure and application of the nano belt in assembling functional stem cell spheres, and belongs to the technical field of biological materials.
Background
The prevalence of degenerative diseases increases year by year with an increase in the aging population. At present, the cause of the diseases is not clear and can not be cured, and the diseases seriously threaten human health and simultaneously cause great economic burden. Among them, neurodegenerative diseases are diseases in which neuronal structures or functions are gradually lost to cause dysfunction, including Parkinson's Disease (PD), alzheimer's Disease (AD), huntington's Disease (HD), amyotrophic lateral sclerosis (ALS, commonly known as evagination syndrome), spinal Muscular Atrophy (SMA), and the like. Bone degenerative diseases including cervical osteoarthritis, proliferative cervical spondylitis, cervical radiculopathy, cervical disc herniation and the like are diseases based on degenerative pathological changes. The key to alleviating and treating degenerative diseases is to repair damaged tissues and construct a normal nerve signaling system.
Stem cells are a class of cells with self-renewing and differentiating potential that, under appropriate circumstances and appropriate signals, differentiate into various types of mature cells, which in turn produce tissue. However, stem cell therapy is also restricted due to factors such as slow differentiation rate and uncertain differentiation direction of stem cells. The current method for regulating stem cell differentiation by adopting biological reagent is also limited by the factors of high price, short effective time and the like. The nano material has adjustable size, simple synthesis method, mature preparation technology and low price, and the application of the nano material in stem cell fate control provides a new solution for tissue regeneration. However, in vitro two-dimensional culture of cells cannot truly simulate the three-dimensional microenvironment in human body, and the cells need to be digested from a traditional culture dish before being transplanted into the body, so that the activity of the cells is reduced and the cells are damaged. The application of the three-dimensional assembled stem cell spheres can effectively maintain cell communication, and is an effective guarantee for in-vivo application.
However, the existing stem cell pellet only has aggregation effect, and can better play the stem cell therapeutic effect after functionalization. Hydroxyapatite has good biocompatibility, is a main component of teeth and bones, and has been studied before project research teams to find that nano hydroxyapatite can be endocytosed by stem cells, stimulate the stem cells and enhance the differentiation of the stem cells. Meanwhile, researches show that the pure stem cell pellet can cause insufficient nutrition in the central part in the culture process, active damage is easy to form, the one-dimensional structure nano material can relieve the problem of difficult nutrition transportation in the stem cell pellet, but the existing one-dimensional structure nano material is mostly used as a medicine carrier, and the one-dimensional structure nano material can not realize the regulation and control effect on the fate of the stem cells by the loaded medicine molecules.
Therefore, the novel one-dimensional nano hydroxyapatite material is developed, and can simultaneously enhance the transportation of stem cell pellet nutrient substances and realize endocytosis to regulate and control stem cell fate, so that the preparation of functionalized stem cell pellets is realized, and new materials and functional cells are provided for efficient tissue repair.
Disclosure of Invention
The invention relates to a preparation method of a one-dimensional multifunctional hydroxyapatite nano belt with a secondary structure and application of the nano belt in assembling functional stem cell spheres.
The one-dimensional multifunctional hydroxyapatite nanobelt with the secondary structure is prepared by a simple method, and is assembled into various functional stem cell spheres by culturing and endocytosis of the stem cell spheres, so that the degenerative diseases can be relieved and treated.
In order to achieve the above purpose, the invention is realized by the following technical scheme:
the first object of the invention is to provide a preparation method of a one-dimensional multifunctional hydroxyapatite nano belt with a secondary structure.
The preparation method of the one-dimensional multifunctional hydroxyapatite nano belt with the secondary structure comprises the following steps:
(1) NaOH and Fe 3 O 4 、CaCl 2 And NaH 2 PO 4 ·2H 2 Adding O into the mixed solution of water and ethanol, and uniformly mixing and stirring to obtain a mixture;
(2) Transferring the mixture into a closed reactor, reacting for 36-72 hours at 120-250 ℃, and naturally cooling to room temperature;
(3) Dispersing the product obtained in the step (2) in cyclohexane, centrifuging and cleaning to obtain a hydroxyapatite nano belt;
(4) Adding Tris-HCl solution into the hydroxyapatite nano belt obtained in the step (3), stirring uniformly, then adding dopamine, stirring, centrifuging the product, and washing with water to obtain the multifunctional hydroxyapatite nano belt.
According to a preferred embodiment of the invention, in step (1), naOH and CaCl 2 The mass ratio of (1-10): (1-2)。
According to a preferred embodiment of the invention, in step (1), caCl 2 With aH 2 The mass ratio of the PO4 & 2H2O is (1-2): (2-3).
According to a preferred embodiment of the invention, in step (1), fe in the mixture 3 O 4 Accounting for 5-30% of the total mass of the mixture.
Further preferably, in step (1), fe in the mixture 3 O 4 Accounting for 10 percent of the total mass of the mixture.
According to the invention, in the step (1), the mixed solution of water and ethanol has the volume ratio of 2:1.
according to a preferred embodiment of the invention, in step (1), caCl 2 The mass ratio of the mixed solution of water and ethanol is (2-6): 1.
according to the invention, in the step (2), after reacting for 48 hours at 200 ℃, naturally cooling to room temperature;
according to a preferred embodiment of the present invention, in step (4), the Tris-HCl solution is 10mM Tris-HCl solution having a pH of=8.5.
According to the invention, in the step (4), the mass ratio of the hydroxyapatite nano belt to the Tris-HCl solution is 1: (8-15).
According to the invention, in the step (4), the mass ratio of the hydroxyapatite nano band to the dopamine is (1-2): (1-2).
The one-dimensional multifunctional hydroxyapatite nano belt with the secondary structure is prepared by the method, has a multi-layer strapped structure, is bifurcated at the end face, has the length of more than or equal to 1 micron and has the width of 8-12nm.
The preparation of the one-dimensional multifunctional hydroxyapatite nano belt with the secondary structure can also be carried out without adding Fe 3 O 4 The one-dimensional hydroxyapatite nano belt with a secondary structure and no magnetism is obtained.
The application of the one-dimensional multifunctional hydroxyapatite nano belt with the secondary structure is used for assembling the functionalized stem cell sphere, and comprises the following steps:
1) Mixing the multifunctional hydroxyapatite nano belt with stem cells, and centrifugally gathering the mixture into balls;
2) And inoculating the centrifugate into a stem cell culture medium to culture and endocytose the stem cells, thereby obtaining the functionalized stem cell pellet.
According to the invention, in step 1), the centrifugation speed is 400-700rpm and the centrifugation time is 4-6min.
According to a preferred embodiment of the present invention, in step 1), the stem cells are neural stem cells, bone marrow mesenchymal stem cells or adipose mesenchymal stem cells.
According to the preferred embodiment of the present invention, in step 2), the concentration of the multifunctional hydroxyapatite nanoribbon in the system is 0.2-500 μg/ml after the multifunctional hydroxyapatite nanoribbon is added.
According to the preferred embodiment of the present invention, in step 2), the concentration of the multifunctional hydroxyapatite nanoribbon in the system is 1-300 μg/ml after the multifunctional hydroxyapatite nanoribbon is added.
According to the preferred embodiment of the present invention, in step 2), the concentration of the multifunctional hydroxyapatite nanoribbon in the system is 20-200 μg/ml after the multifunctional hydroxyapatite nanoribbon is added.
According to a preferred embodiment of the invention, in step 2), the amount of stem cells in the system is 1-7 x 10 7 And each ml.
According to a preferred embodiment of the present invention, in step 2), when the stem cells are neural stem cells, the stem cell culture medium is a neural stem cell differentiation medium, and the neural stem cell differentiation medium includes a basal medium, a Neurobasal medium, gibco B-27 neural cell culture serum-free additive having a volume concentration of 1-3%, fetal bovine serum FBS having a volume concentration of 1-2%, and a diabody having a volume concentration of 1-2%.
According to a preferred embodiment of the present invention, in step 2), when the stem cells are bone marrow mesenchymal stem cells, the stem cell culture medium is a bone marrow mesenchymal stem cell culture medium comprising an α basal medium, fetal bovine serum FBS at a volume concentration of 1-2% and a diabody at a volume concentration of 1-2%.
According to a preferred embodiment of the present invention, in step 2), when the stem cells are adipose-derived mesenchymal stem cells, the stem cell culture medium is adipose-derived mesenchymal stem cell culture medium comprising an α -basal medium, fetal bovine serum FBS at a volume concentration of 1-2% and a diabody at a volume concentration of 1-2%.
According to a preferred embodiment of the invention, in step 2), the stem cells are cultured and endocytosed by incubating the system in an incubator at 36-37℃for 1-14 days.
The stem cells of the invention may be isolated from an embryo or adult brain according to known methods. Alternatively, stem cells may be purchased from the market, or may be cultured by any conventional method known in the art. There is no particular limitation on the above. In the examples section that follows, the method of stem cell extraction is described in detail.
The invention has the technical characteristics and advantages that:
1. the hydroxyapatite nano-belt obtained by the invention has a multi-layer stack shape and is bifurcated at the end face, is a one-dimensional multifunctional hydroxyapatite nano-belt with a secondary structure, the length of the nano-belt is more than or equal to 1 micron, the width of the nano-belt is 8-12nm, the multi-layer stack structure is convenient for transporting nutrition into stem cell spheres, the end face is easy to diverge, the endocytosis of cells is facilitated, and the interaction between cells and materials is promoted.
2. The hydroxyapatite nanobelt obtained by the invention contains magnetic nanoparticles, so that the stem cell spheres can be better fixed on an affected part; the poly-dopamine coating is externally applied to strengthen cell adhesion, so that the poly-dopamine nano-belt is a multifunctional hydroxyapatite nano-belt; the multi-layer stack structure of the nanoribbon helps the inside of the stem cell pellet to transport nutrients, oxygen, etc.
3. The one-dimensional multifunctional hydroxyapatite nano-belt with the secondary structure is used for assembling the functionalized stem cell spheres, has a high nerve differentiation speed and a remarkable neuron direction differentiation trend, can generate more mature neurons in a shorter time, and can maximally improve the nerve tissue repair efficiency; the mesenchymal stem cells modified by the multifunctional hydroxyapatite nano belt have a relatively high osteogenic differentiation speed.
4. The one-dimensional multifunctional hydroxyapatite nano belt with the secondary structure is used for assembling the functionalized stem cell spheres, and the assembling method is simple in preparation, long in aging, low in immunogenicity, low in storage and transportation difficulty and wide in application prospect.
Drawings
Figure 1 is a schematic diagram of the synthesis of a multifunctional nanoribbon,
fig. 2 is a TEM image and a schematic view of a hydroxyapatite nanobelt with a secondary structure, a is a TEM image, and b is a schematic view;
FIG. 3 is a TEM image of different hydroxyapatite nanoribbons, a is hydroxyapatite nanoribbon 1, b is hydroxyapatite nanoribbon 2, c is hydroxyapatite nanoribbon 3, d is example 1, a1, b1, c1, d1 are TEM images of a, b, c, d nanoribbons laid across, respectively, all on a scale of 50nm.
FIG. 4 shows the viable and dead staining patterns of cell pellets and single cells around one week, A being single cells and B being cell pellets.
FIG. 5 shows different live and dead staining patterns of neural stem cells, assembled by NS neural stem cells, assembled by nanobelts 1, FH-NS, assembled by nanobelts 2, HP-NS, assembled by nanobelts 3, assembled by FHP-NS,
FIG. 6 is a chart showing different treatments of live and dead staining of mesenchymal stem cells, wherein Control is a pellet assembled by mesenchymal stem cells, NB is a pellet assembled by nano-ribbon 1, mag-NB is a pellet assembled by nano-ribbon 2, adh-NB is a pellet assembled by nano-ribbon 3, mag+Adh-NB is a pellet assembled by example 1,
FIG. 7 shows different live and dead staining patterns of adipose-derived mesenchymal stem cells, wherein Control is a pellet assembled by adipose-derived mesenchymal stem cells, NB is a pellet assembled by nanoribbon 1, mag-NB is a pellet assembled by nanoribbon 2, adh-NB is a pellet assembled by nanoribbon 3, mag+Adh-NB is a pellet assembled by example 1,
FIG. 8 is a graph of global hypoxia factor and apoptosis staining of different treated neural stem cells; NS is a neural stem cell assembled stem cell pellet, FHP-NS is the neural stem cell pellet assembled in example 1;
FIG. 9 is a graph showing differential treatment of mesenchymal stem cell hypoxic factors and apoptosis staining; control mesenchymal stem cell assembled cell pellet, NW is stem cell pellet assembled in example 1;
FIG. 10 is a graph showing the neural differentiation promoting gene expression of neural stem cells, neural stem cell spheres assembled by hydroxyapatite nanoribbons 1, and neural stem cell spheres assembled by one-dimensional hydroxyapatite nanoribbons of nonmagnetic secondary structure;
fig. 11 is a graph showing the expression of neural differentiation protein promoted by neural stem cells, neural stem cell spheres assembled by hydroxyapatite nanoribbons 1, and neural stem cell spheres assembled by one-dimensional hydroxyapatite nanoribbons of nonmagnetic secondary structure.
Fig. 12 is a diagram showing the gene expression of bone differentiation promoting effect of bone mesenchymal stem cell single cells, bone mesenchymal stem cell spheres assembled by hydroxyapatite nanoribbon 1, and bone mesenchymal stem cell spheres assembled by one-dimensional hydroxyapatite nanoribbon with nonmagnetic secondary structure.
Fig. 13 is a graph showing bone differentiation promoting protein expression of bone marrow mesenchymal stem cell single cells, bone marrow mesenchymal stem cell spheres assembled by hydroxyapatite nanoribbon 1, and bone marrow mesenchymal stem cell spheres assembled by non-magnetic two-dimensional hydroxyapatite nanoribbon.
Detailed Description
The following examples are given to further illustrate the present invention, but the embodiments of the present invention are not limited to these.
In the examples: the neural stem cells were extracted as follows:
1. the day before the extraction experiment, the surgical bending trays 2, the ophthalmic scissors 1, the hemostatic forceps 4, the surgical scissors 3, the straight forceps 3, the elbow forceps 3 and the 200-mesh filter screen are subjected to high-pressure sterilization and are placed in a 60-DEG oven for drying for standby. The laboratory animals were supplied by the laboratory animal center at the university of Shandong, medical college.
2. 1 is takenC57 mice pregnant for 14 days were sacrificed by cervical removal after 1ml of 10% chloral hydrate was injected for anesthesia, then sterilized by soaking in 75% alcohol for 15min, abdominal skin and muscles were cut off in a biosafety cabinet, and embryos were immediately removed and placed in 4 ℃ pre-chilled PBS buffer. Removing embryo head, dissecting meninges and blood vessels under 10 times dissecting scope, removing cortex parts of left and right brain, placing into PBS buffer solution pre-cooled at 4deg.C, and cutting into pieces of about 0.1cm 3 Repeatedly blowing and beating fragments with the size, filtering by a 200-mesh filter screen, taking filtrate, adding a pre-prepared serum-free culture medium, inoculating into a culture flask, and then placing into a culture box with the temperature of 37 ℃ and the concentration of 5% CO2 for culture. 1ml of a new proliferation medium special for serum-free neural stem cells is added every day, the growth state of the cells is observed by a conventional inverted microscope, and the cells are cultured for 3 to 5 days and then passaged. The cell morphology was directly observed with bright field or after staining the cytoskeleton and nuclei was observed with a fluorescence microscope.
The extraction steps of the mesenchymal stem cells are as follows:
1. laboratory tools and Wistar rats (4-5 weeks old, male and female are not limited) are prepared in advance, and laboratory rooms, sterile operation tables and the culture tools are sterilized in advance.
2. After dislocation of Wistar rats, the two-sided femur and tibia were separated under aseptic conditions on an aseptic console after 5min of 75% ethanol soaking, and the two-sided femur and tibia were separated at a temperature of 50mm containing physiological saline 2 The perifemoral muscle tissue was removed from the glass plate, the epiphyseal ends on both sides including the epiphyseal plate were cut off, a proper amount of complete medium containing 10% FBS was withdrawn with a 10ml syringe (equipped with a 4.5-gauge needle) to flush the cavity, the cells were flushed into a flask, the cell suspension was repeatedly blown with a 7-gauge needle and a 4.5-gauge needle (or 21, 23, 25-gauge needles in order) to prepare a single cell suspension, the cell suspension was collected after centrifugation at 1000rpm for 5min, the cells were thoroughly washed (500 g centrifugation for 5min or 180g centrifugation for 10 min) 2 times with an equal amount of serum-free medium or Phosphate Buffer (PBS), the supernatant was removed, and the cells were suspended with 10% FBS.
The extraction steps of the adipose-derived mesenchymal stem cells are as follows:
taking subcutaneous adipose tissue of rat belly, firstly clamping the adipose tissue by using scissors forceps, and then taking the adipose tissueClamping with forceps, and rinsing with PBS; placing adipose tissue in a small amount of PBS; repeatedly shearing into pieces of about 1mm 3 A fine tissue mass; then 1mg/ml collagenase I digestion solution, twice the volume of the tissue mass, was added and transferred to a 15ml centrifuge tube, and then the tube was placed in a shaking table at 37℃for shaking digestion for 1 hour, after which the digestion was stopped by adding an equal volume of basal medium, and the cells were settled for 5min at 1000 rpm. The supernatant and the suspended adipose tissue were discarded, and the adipose-derived mesenchymal stem cells complete medium was added.
Example 1
The preparation method of the one-dimensional multifunctional hydroxyapatite nano belt with the secondary structure comprises the following steps:
(1) 0.5g of NaOH and Fe 3 O 4 、176mg CaCl 2 And 230mg NaH 2 PO 4 ·2H 2 Adding O into 80ml of mixed solution of water and ethanol (the volume ratio of water to ethanol is 2:1), mixing and stirring uniformly to obtain a mixture, wherein Fe is contained in the mixture 3 O 4 Accounting for 10 percent of the total mass of the mixture;
(2) Transferring the mixture into a closed reactor, reacting for 48 hours at 200 ℃, and naturally cooling to room temperature;
(3) Dispersing the product obtained in the step (2) in cyclohexane, centrifuging, and then washing with cyclohexane and ethanol for three times to obtain a hydroxyapatite nano belt;
(4) Adding 10mM Tris-HCl solution with PH=8.5 into the hydroxyapatite nano belt obtained in the step (3), stirring uniformly, adding dopamine, stirring for 24 hours, centrifuging the product, and washing with water to obtain the multifunctional hydroxyapatite nano belt; the mass ratio of the hydroxyapatite nano belt to the Tris-HCl solution is 1:12, the mass ratio of the hydroxyapatite nano band to the dopamine is 1:1.
the multifunctional nanoribbon synthesis schematic diagram is shown in figure 1.
The TEM with the secondary structure of the prepared multifunctional hydroxyapatite nano belt is shown in figure 2, and the synthesized multifunctional hydroxyapatite nano belt has a multi-layer haystack structure and a bifurcated end face as can be seen from figure 2.
Example 2
The preparation method as described in example 1 is different in that:
in step (1), 0.3g of NaOH and Fe 3 O 4 、120mg CaCl 2 And 210mg NaH 2 PO 4 ·2H 2 Adding O into 80ml of mixed solution of water and ethanol (the volume ratio of water to ethanol is 2:1), mixing and stirring uniformly to obtain a mixture, wherein Fe is contained in the mixture 3 O 4 8% of the total mass of the mixture, the other steps were carried out as in example 1.
Example 3
The preparation method as described in example 1 is different in that:
in step (1), 0.7g of NaOH and Fe 3 O 4 、180mg CaCl 2 And 270mg NaH 2 PO 4 ·2H 2 Adding O into 80ml of mixed solution of water and ethanol (the volume ratio of water to ethanol is 2:1), mixing and stirring uniformly to obtain a mixture, wherein Fe is contained in the mixture 3 O 4 16% of the total mass of the mixture, the other steps were carried out as in example 1.
Example 4
The application of the two-dimensional multifunctional hydroxyapatite nanobelt with the secondary structure in the embodiment 1 is used for assembling the functionalized stem cell ball and comprises the following steps:
1) Mixing the multifunctional hydroxyapatite nano belt with the neural stem cells, centrifuging for 5min at 500rpm, and agglomerating into balls;
2) Inoculating the centrifugate into neural stem cell differentiation medium, adding multifunctional hydroxyapatite nanobelt, wherein the concentration of the multifunctional hydroxyapatite nanobelt in the system is 100 μg/ml, and the amount of stem cells in the system is 10 7 And each ml. Culturing neural stem cells and endocytosis by incubating the culture system in an incubator at 36-37 ℃ for 1-7 days, and assembling the stem cells into stem cell spheres.
The neural stem cell differentiation medium comprises a basic medium, namely a Neurobasal medium, wherein the Gibco B-27 neural cell culture serum-free additive with the volume concentration of 1-3 percent, fetal bovine serum FBS with the volume concentration of 1-2 percent and diabody with the volume concentration of 1-2 percent.
Example 5
The application of the two-dimensional multifunctional hydroxyapatite nanobelt with the secondary structure in the embodiment 1 is used for assembling the functionalized stem cell ball and comprises the following steps:
1) Mixing the multifunctional hydroxyapatite nano belt with bone marrow mesenchymal stem cells, centrifuging at 500rpm for 5min, and agglomerating into balls;
2) Inoculating the centrifugate into bone marrow mesenchymal stem cell culture medium, adding multifunctional hydroxyapatite nanobelt, wherein the concentration of the multifunctional hydroxyapatite nanobelt in the system is 150 μg/ml, and the amount of stem cells in the system is 10 7 And each ml. Culturing in 36-37deg.C incubator for 1-14 days, culturing and endocytosis of bone marrow mesenchymal stem cells, assembling bone marrow mesenchymal stem cells into stem cell spheres, and shaking on 25 deg.C 40rpm shaker for 1 week.
The bone marrow mesenchymal stem cell culture medium comprises an alpha basal culture medium, fetal bovine serum FBS with the volume concentration of 1-2% and diabody with the volume concentration of 1-2%.
Example 6
The application of the two-dimensional multifunctional hydroxyapatite nanobelt with the secondary structure in the embodiment 1 is used for assembling the functionalized stem cell ball and comprises the following steps:
1) Mixing the multifunctional hydroxyapatite nano belt with the adipose-derived mesenchymal stem cells, centrifuging at 500rpm for 5min, and agglomerating into balls;
2) Inoculating the centrifugate into a adipose-derived mesenchymal stem cell culture medium, adding multifunctional hydroxyapatite nanoribbon, wherein the concentration of the multifunctional hydroxyapatite nanoribbon in the system is 200 mug/ml, and the amount of stem cells in the system is 10 7 And each ml. The culture system is placed in an incubator at 36-37 ℃ for 1-14 days for culturing and endocytosis of the adipose-derived mesenchymal stem cells, and the adipose-derived mesenchymal stem cells are assembled into stem cell spheres.
The adipose-derived mesenchymal stem cell culture medium comprises an alpha basal medium, fetal bovine serum FBS with the volume concentration of 1-2% and diabody with the volume concentration of 1-2%.
Comparative example 1
The preparation method of the two-dimensional multifunctional hydroxyapatite nano belt with the secondary structure is the same as that of the embodiment 1, and is different in that:
(1) 0.5g NaOH and 176mg CaCl were added 2 And 230mg NaH 2 PO 4 ·2H 2 Adding O into 80ml of mixed solution of water and ethanol (the volume ratio of water to ethanol is 2:1), and uniformly mixing and stirring to obtain a mixture;
(2) Transferring the mixture into a closed reactor, reacting for 48 hours at 200 ℃, naturally cooling to room temperature, dispersing the obtained product in cyclohexane, centrifuging, and then washing with cyclohexane and ethanol for three times to obtain the hydroxyapatite nanobelt 1.
Comparative example 2
The preparation method of the two-dimensional multifunctional hydroxyapatite nano belt with the secondary structure is the same as that of the embodiment 1, and is different in that:
(1) 0.5g of NaOH and Fe 3 O 4 、176mg CaCl 2 And 230mg NaH 2 PO 4 ·2H 2 Adding O into 80ml of mixed solution of water and ethanol (the volume ratio of water to ethanol is 2:1), mixing and stirring uniformly to obtain a mixture, wherein Fe is contained in the mixture 3 O 4 Accounting for 10 percent of the total mass of the mixture;
(2) Transferring the mixture into a closed reactor, reacting for 48 hours at 200 ℃, and naturally cooling to room temperature;
(3) Dispersing the product obtained in the step (2) in cyclohexane, centrifuging, and then washing with cyclohexane and ethanol for three times to obtain the hydroxyapatite nano belt 2.
Comparative example 3
The preparation method of the two-dimensional multifunctional hydroxyapatite nano belt with the secondary structure is the same as that of the embodiment 1, and is different in that:
(1) 0.5g NaOH and 176mg CaCl were added 2 And 230mg NaH 2 PO 4 ·2H 2 Adding O into 80ml of mixed solution of water and ethanol (the volume ratio of water to ethanol is 2:1), mixing and stirring uniformly to obtain a mixture,
(2) Transferring the mixture into a closed reactor, reacting for 48 hours at 200 ℃, and naturally cooling to room temperature;
(3) Dispersing the product obtained in the step (2) in cyclohexane, centrifuging, and then washing with cyclohexane and ethanol for three times sequentially to obtain the hydroxyapatite nano belt.
(4) Adding 10mM Tris-HCl solution with PH=8.5 into the hydroxyapatite nano belt obtained in the step (3), stirring uniformly, adding dopamine, stirring for 24 hours, centrifuging the product, and washing with water to obtain the hydroxyapatite nano belt 3; the mass ratio of the hydroxyapatite nano belt to the Tris-HCl solution is 1:12, the mass ratio of the hydroxyapatite nano band to the dopamine is 1:1.
experimental example:
1. the TEM of the two-dimensional multifunctional hydroxyapatite nanobelts of the two-dimensional structure obtained in the comparative examples 1-3 and the two-dimensional multifunctional hydroxyapatite nanobelt of the example 1 are shown in the figure 3, and as can be seen from the figure 3, the two-dimensional multifunctional hydroxyapatite nanobelts of the multiple example 1 have the length of more than 1 micrometer and the width of about 10nm, and the multilayer haystack structure of the multifunctional hydroxyapatite nanobelts takes the role of a nutrition conveying belt.
2. a, inoculating neural stem cells into a neural stem cell differentiation culture medium, and shaking on a shaking table at 40rpm at 25 ℃ for 1 week, wherein the amount of stem cells in the system is 10 7 Individual/ml; the neural stem cells were obtained as single cells (other single cells were performed as such).
b. Centrifuging neural stem cells at 500rpm for 5min, agglomerating into spheres, inoculating into neural stem cell differentiation medium, and shaking on shaking table at 25deg.C and 40rpm for 1 week to obtain stem cells with an amount of 10 7 Individual/ml; neural stem cell spheres (other stem cell spheres are performed accordingly) were obtained.
Live-dead staining of single cells and neural stem cell spheres before and after one week without assembly is shown in fig. 4.
3. The hydroxyapatite nanobelts 1-3 and the nanobelt of the embodiment 1 are cultured and endocytosed for 3 days according to the method of the embodiment 4, the neural stem cells are assembled into stem cell spheres, and the stem cell spheres are compared with unassembled neural stem cell spheres, and a live dying color chart is shown in fig. 5, so that the secondary structure one-dimensional multifunctional hydroxyapatite nanobelt of the invention has good biocompatibility.
4. The hydroxyapatite nanobelts 1-3 and the nanobelt of the embodiment 1 are used for culturing and endocytosis of bone marrow mesenchymal stem cells for 3 days according to the method of the embodiment 5, the bone marrow mesenchymal stem cells are assembled into bone marrow mesenchymal stem cell spheres, and the bone marrow mesenchymal stem cell spheres are compared with bone marrow mesenchymal stem cell spheres which are not assembled, a live dying staining chart is shown in fig. 6, and the secondary structure one-dimensional multifunctional hydroxyapatite nanobelt disclosed by the invention is good in biocompatibility.
5. The hydroxyapatite nanobelts 1-3 and the nanobelt of the embodiment 1 are used for culturing and endocytosis of the adipose-derived mesenchymal stem cells for 3 days according to the method of the embodiment 6, the adipose-derived mesenchymal stem cells are assembled into adipose-derived mesenchymal stem cell bodies, and the adipose-derived mesenchymal stem cell bodies are compared with unassembled adipose-derived mesenchymal stem cell spheres, and a living dying color chart is shown in fig. 7, so that the two-level structure one-dimensional multifunctional hydroxyapatite nanobelt of the invention has good biocompatibility.
6. To verify the nutrition delivery effect of the multifunctional nanoribbon, the nanoribbon of example 1 was cultured and endocytosed for 3 days as in example 4, the neural stem cells were assembled into stem cell spheres, and hypoxia factor and apoptosis staining were performed in comparison with the neural stem cell spheres not assembled, as shown in fig. 8.
To verify the nutrition delivery effect of the multifunctional nanobelt, the nanobelt of example 1 was cultured and endocytosed for 3 days as in example 5, bone marrow mesenchymal stem cells were assembled into stem cell spheres, and hypoxia factor and apoptosis staining were performed in comparison with bone marrow mesenchymal stem cell spheres not assembled, as shown in fig. 9.
In summary, fig. 8 and fig. 9 illustrate that the nanoribbon of the present invention can transport nutrition and oxygen inside the stem cell pellet, and that pure stem cell pellets can cause insufficient nutrition in the central portion during the culturing process, thus easily forming active damage.
7. Nerve differentiation promoting effect of multifunctional nanoribbon:
for in vitro validation, the preparation was performed as in example 1, without adding Fe 3 O 4 To obtain the one-dimensional hydroxyapatite nano-with non-magnetic secondary structureA rice belt.
The gene expression and protein expression of the neural stem single cells, the neural stem cell spheres assembled by the hydroxyapatite nano-belt 1 and the neural stem cell spheres assembled by the non-magnetic two-dimensional hydroxyapatite nano-belt are shown in figures 10 and 11.
8. Nerve differentiation promoting effect of multifunctional nanoribbon:
for in vitro validation, the preparation was performed as in example 1, without adding Fe 3 O 4 The one-dimensional hydroxyapatite nano belt with a secondary structure and no magnetism is obtained.
The bone marrow mesenchymal stem cells assembled by the bone marrow mesenchymal stem cell spheres, the bone marrow mesenchymal stem cell spheres assembled by the hydroxyapatite nano belt 1 and the bone marrow mesenchymal stem cell spheres assembled by the non-magnetic two-dimensional hydroxyapatite nano belt with the two-dimensional structure are shown in figures 12 and 13.
In conclusion, the two-level structure one-dimensional multifunctional hydroxyapatite nanobelt is used for assembling the functionalized stem cell sphere, has a high nerve differentiation speed and a remarkable neuron direction differentiation trend, can generate more mature neurons in a shorter time, and improves nerve tissue repair efficiency to the greatest extent; the mesenchymal stem cells modified by the multifunctional hydroxyapatite nano belt have a relatively high osteogenic differentiation speed, and can effectively promote tissue regeneration.
Claims (10)
1. The preparation method of the one-dimensional multifunctional hydroxyapatite nano belt with the secondary structure comprises the following steps:
(1) NaOH and Fe 3 O 4 、CaCl 2 And NaH 2 PO 4 ·2H 2 Adding O into the mixed solution of water and ethanol, and uniformly mixing and stirring to obtain a mixture; naOH and CaCl 2 The mass ratio of (1-10): (1-2) CaCl 2 With aH 2 The mass ratio of the PO4 & 2H2O is (1-2): (2-3) in the mixture, fe 3 O 4 Accounting for 5-30% of the total mass of the mixture;
(2) Transferring the mixture into a closed reactor, reacting for 36-72 hours at 120-250 ℃, and naturally cooling to room temperature;
(3) Dispersing the product obtained in the step (2) in cyclohexane, centrifuging and cleaning to obtain a hydroxyapatite nano belt;
(4) Adding Tris-HCl solution into the hydroxyapatite nano belt obtained in the step (3), stirring uniformly, then adding dopamine, stirring, centrifuging the product, and washing with water to obtain the multifunctional hydroxyapatite nano belt.
2. The process according to claim 1, wherein in step (1), fe is contained in the mixture 3 O 4 Accounting for 10 percent of the total mass of the mixture.
3. The method according to claim 1, wherein in the step (1), the volume ratio of the water to the ethanol is 2:1, caCl 2 The mass ratio of the mixed solution of water and ethanol is (2-6): 1.
4. the method according to claim 1, wherein in the step (2), after reacting at 200 ℃ for 48 hours, naturally cooling to room temperature; in the step (4), the Tris-HCl solution is 10mM, the pH=8.5 Tris-HCl solution, and the mass ratio of the hydroxyapatite nano band to the Tris-HCl solution is 1: (8-15), the mass ratio of the hydroxyapatite nano belt to the dopamine is (1-2): (1-2).
5. A one-dimensional multifunctional hydroxyapatite nanobelt with a secondary structure, which is prepared by adopting any one of the methods of claims 1-4, has a multi-layer haystack structure, is bifurcated at the end surface, has a length of 1 micrometer or more and a width of 8-12nm.
6. The use of the two-dimensional multifunctional hydroxyapatite nanobelt with a secondary structure according to claim 5 for assembling functionalized stem cell balls, comprising the steps of:
1) Mixing the multifunctional hydroxyapatite nano belt with stem cells, and centrifugally gathering the mixture into balls;
2) And inoculating the centrifugate into a stem cell culture medium to culture and endocytose the stem cells, thereby obtaining the functionalized stem cell pellet.
7. The use according to claim 6, wherein in step 1) the centrifugation speed is 400-700rpm and the centrifugation time is 4-6min; the stem cells are neural stem cells, bone marrow mesenchymal stem cells or adipose mesenchymal stem cells.
8. The use according to claim 6, wherein in step 2) the concentration of the multifunctional hydroxyapatite nanoribbon in the system is 20-200 μg/ml after adding the multifunctional hydroxyapatite nanoribbon, and the amount of stem cells in the system is 1-7 x 10 7 And each ml.
9. The use according to claim 6, wherein in step 2), when the stem cells are neural stem cells, the stem cell culture medium is a neural stem cell differentiation medium, the neural stem cell differentiation medium comprises a basal medium, a Neurobasal medium, a Gibco B-27 neural cell culture serum-free additive in a volume concentration of 1-3%, a fetal bovine serum FBS in a volume concentration of 1-2%, and a diabody in a volume concentration of 1-2%;
when the stem cells are bone marrow mesenchymal stem cells, the stem cell culture medium is a bone marrow mesenchymal stem cell culture medium, and the bone marrow mesenchymal stem cell culture medium comprises an alpha basal culture medium, fetal bovine serum FBS with the volume concentration of 1-2% and diabody with the volume concentration of 1-2%;
when the stem cells are adipose-derived mesenchymal stem cells, the stem cell culture medium is adipose-derived mesenchymal stem cell culture medium, and the adipose-derived mesenchymal stem cell culture medium comprises an alpha basal culture medium, fetal bovine serum FBS with the volume concentration of 1-2% and diabody with the volume concentration of 1-2%.
10. The use according to claim 6, wherein in step 2) the stem cells are cultured and endocytosed by incubating the system in an incubator at 36-37 ℃ for 1-14 days.
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