CN111195370B - High-magnesium microenvironment bone marrow stem cell microsphere carrier and preparation method and application thereof - Google Patents

High-magnesium microenvironment bone marrow stem cell microsphere carrier and preparation method and application thereof Download PDF

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CN111195370B
CN111195370B CN201811366500.1A CN201811366500A CN111195370B CN 111195370 B CN111195370 B CN 111195370B CN 201811366500 A CN201811366500 A CN 201811366500A CN 111195370 B CN111195370 B CN 111195370B
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magnesium
stem cells
microsphere carrier
cell
microenvironment
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CN111195370A (en
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蒋欣泉
张文杰
林思涵
杨光正
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Ninth Peoples Hospital Shanghai Jiaotong University School of Medicine
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Abstract

The invention provides a high-magnesium microenvironment bone marrow stem cell microsphere carrier, a preparation method and an application thereof, wherein magnesium ions are compounded into the microsphere carrier to form a local high-magnesium environment, the size of the microsphere carrier is within 500 mu m, and the concentration of the magnesium ions is 2.5mM-10mM. The invention provides a concept and a preparation method for constructing a local high-magnesium microenvironment stem cell microsphere carrier, on one hand, when the concentration of screened magnesium ions is 2.5mM-5mM, the osteogenesis induction effect is optimal; secondly, the diameter of the prepared stem cell microsphere carrier is within 500 mu m, so that the load rate and the survival rate of stem cells are effectively improved, the osteogenic differentiation of the loaded stem cells is promoted by utilizing a local high-magnesium environment, and the defects of high cost, difficult control of slow release and the like caused by using protein factors are reduced; finally, when the prepared high-magnesium microenvironment stem cell microsphere carrier is used for bone defect regeneration repair, the number of new bones in the defect area is obviously increased, and the repair effect is obviously improved.

Description

High-magnesium microenvironment bone marrow stem cell microsphere carrier and preparation method and application thereof
Technical Field
The invention belongs to the field of tissue engineering and regenerative medicine, and particularly relates to a high-magnesium microenvironment bone marrow stem cell microsphere carrier, and a preparation method and application thereof.
Background
Stem cells have self-renewal ability and multipotential differentiation ability, and theoretically can be expanded in vitro to a sufficient number and induced to differentiate into specific adult cells for tissue regeneration treatment. Direct loading of cells onto scaffold materials is a common method, but due to the influence of material pore size and specific surface area, the number of transplanted cells is significantly limited: too small pore diameter of the porous scaffold material can limit the cells from permeating into the deep part of the scaffold, and the specific surface area is reduced along with the expansion of the pore diameter, thereby influencing the loading quantity of the cells. The microspheres are used as a stem cell carrier, compared with the traditional support, the microsphere has the advantages of small size, easy nutrition penetration and large specific surface area and large cell loading capacity, and meanwhile, the microsphere 3D cultured stem cells effectively maintain the cell dryness, provide a stable local environment for the stem cells, effectively resist the immune rejection of a receptor and the like, and have more advantages in tissue regeneration application.
Magnesium is a major element of human body and is indispensable in bone growth and development, and the function of promoting osteogenesis by magnesium ions has been reported in many studies. In order to further improve the regeneration and repair effect, magnesium ions with osteogenic differentiation induction capability are compounded into microspheres, high-magnesium microenvironment microspheres are constructed, and stem cells are delivered for bone regeneration at the same time, and the research on the aspect is not reported in documents.
Disclosure of Invention
The invention aims to provide a bone marrow stem cell microsphere carrier with a high-magnesium microenvironment.
The second purpose of the invention is to provide a preparation method of the bone marrow stem cell microsphere carrier with the high magnesium microenvironment.
The third purpose of the invention is to provide the application of the bone marrow stem cell microsphere carrier with high magnesium microenvironment in bone regeneration.
In order to realize the first purpose of the invention, the invention discloses the following technical scheme: the high-magnesium microenvironment bone marrow stem cell microsphere carrier is characterized in that magnesium ions are compounded into the microsphere carrier to form a local high-magnesium environment, the size of the microsphere carrier is 100-500 mu m, and the concentration of the magnesium ions is 2.5-10 mM.
Preferably, the magnesium ion concentration is in the range of 2.5mM to 5mM, which is optimal for osteoinduction.
As a preferred scheme, the density of the stem cells loaded on the microsphere carrier is 5 x 10 6 -10 7 /ml。
In order to realize the second purpose of the invention, the invention discloses the following technical scheme: a preparation method of a high-magnesium microenvironment bone marrow stem cell microsphere carrier is characterized by comprising the following steps:
(1) Obtaining and culturing osteogenesis related stem cells;
(2) Digesting and centrifuging the stem cells obtained in the step (1), then re-suspending the stem cells by using a high-magnesium culture solution, mixing the obtained cell suspension with gel, and dripping the gel into a cross-linking agent by using a syringe to obtain the cell-loaded microspheres, wherein the diameter of the cell-loaded microspheres is 100-500 microns, and the concentration of magnesium ions is 2.5-10 mM.
As a preferable embodiment, the magnesium ion concentration selected in step (2) is in the range of 2.5mM to 5mM, which is the most excellent for the osteogenesis inducing effect.
As a preferable scheme, the step (2) uses sodium alginate gel, and the final concentration of the sodium alginate gel after gelling is 1% -2%.
As a preferred variant, the crosslinking agent used is CaCl 2 ，SrCl 2 Or BaCl 2 To (3) is provided.
Preferably, the inner diameter of the syringe needle is 150 μm to 180 μm, and the cell-gel mixture is dropped using a micro-applicator so that the drop volume is within 0.4. Mu.L and the needle is within 1mm from the surface of the crosslinking agent.
In order to achieve the third purpose of the invention, the invention discloses the following technical scheme: the application of the high-magnesium microenvironment bone marrow stem cell microsphere carrier in bone regeneration. The hydrogel stem cell microsphere carrier can be injected alone or matched with a traditional bracket to repair bone defects, and meanwhile, the injection mode can reduce surgical wounds and avoid complications such as wound infection and the like.
The invention evaluates the influence of magnesium ions with different concentrations on the activity of the bone marrow stem cells, and screens out the concentration range of the magnesium ions suitable for the survival of the cells; on the basis, the influence of magnesium ions on the osteogenic differentiation of stem cells is detected, and the magnesium ion concentration range with the best osteoinduction effect is optimized; preparing a high-magnesium microenvironment bone marrow stem cell microsphere carrier, evaluating the survival rate of stem cells and the osteogenic differentiation effect, and screening the microsphere size with the best cell survival rate and the concentration of magnesium ions for promoting osteogenic differentiation of the stem cells; the prepared bone marrow stem cell microsphere carrier with high magnesium microenvironment is transplanted in vivo to observe the bone regeneration effect. The research result shows that: (1) The magnesium ion concentration is within 20mM, has no obvious cytotoxicity, and has certain effect of promoting cell proliferation (2) when the magnesium ion concentration is within 10mM, the osteogenesis induction effect is realized; when the concentration of magnesium ions is 2.5mM-5mM, the best osteogenic differentiation effect of the stem cells is induced, (3) the stem cell-loaded sodium alginate microspheres with the diameter within 1mM are successfully prepared, and the cell survival rate is the highest when the diameter of the microspheres is 500 mu m; (4) The microsphere local magnesium ion concentration is 2.5mM-5mM, the osteogenic differentiation of the stem cells can be promoted, and the microsphere has the effect of promoting cell proliferation (5), and the high-magnesium microenvironment stem cell microsphere can obviously promote the bone defect repair.
The invention has the advantages that: the invention provides a concept and a preparation method for constructing a local high-magnesium microenvironment stem cell microsphere carrier, on one hand, magnesium ions screened out with the concentration within 20mM have no toxicity to cells and have the effect of promoting cell proliferation, the magnesium ion concentration has the osteogenesis induction effect when being 2.5mM-10mM, and the osteogenesis induction effect is optimal when the magnesium ion concentration is 2.5mM-5 mM; secondly, the diameter of the prepared stem cell microsphere carrier is 100-500 μm, so that the load rate and the survival rate of stem cells are effectively improved, and the defects of high cost, difficult slow release and the like caused by using protein factors are reduced by promoting osteogenic differentiation of the loaded stem cells in a local high-magnesium environment; finally, when the prepared high-magnesium microenvironment stem cell microsphere carrier is used for bone defect regeneration repair, the number of new bones in the defect area is obviously increased, and the repair effect is obviously improved. The invention does not need special, complex and expensive equipment, has simple operation flow and is beneficial to popularization and use.
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FIG. 1: effect of different concentrations of magnesium ions on stem cell activity. (a) Half maximal Inhibitory Concentration (IC) of magnesium ions 50 ) Curve, IC 50 =65.91mM; (b) MTT method for detecting the influence of different concentrations of magnesium ions on cell proliferation (ridiculo, p)<0.01)。
FIG. 2: the magnesium ions with different concentrations have the effect of inducing the osteogenic differentiation of stem cells. (a) ALP staining results; (b) an ALP semiquantitative result ({ major:, p < 0.01); (c) Weterblot detects the expression of Osteocalcin (OCN) of stem cells; and (d) stem cell OCN immunofluorescence staining results.
FIG. 3: the magnesium ions with different concentrations induce the expression change of a stem cell magnesium ion channel MagT 1. (a) stem cell MagT1qPCR results after osteogenic induction; (b) Performing immunofluorescence double staining on stem cells MagT1 and OCN after osteogenesis induction; (c) Performing MagT1qPCR (quantitative polymerase chain reaction) on stem cells induced by magnesium ions with different concentrations; (d) And performing immunofluorescence staining on the stem cells MagT1 after induction by different magnesium ions.
FIG. 4: and (3) performing live-dead staining results of stem cells in microspheres with different sizes after 3 days of culture (green represents live cells, and red represents dead cells).
FIG. 5: effect of high magnesium microenvironment on stem cell activity within microspheres. (a) Culturing for 1 day and 3 days to obtain live and dead staining results of stem cells in the microspheres; and (b) detecting the survival condition of the stem cells in the microspheres by a CCK8 method.
FIG. 6: and detecting the proliferation activity of the stem cells in the high-magnesium micro-environment microspheres by EdU staining. (a) After the high-magnesium microenvironment stem cell microspheres are frozen and sliced, an EdU staining result (red is an EdU positive cell, blue is a cell nucleus, and pink is an EdU true positive cell) is obtained; (b) The rate of proliferation of stem cells in the microsphere stem cell vector (articulator: (p < 0.01)).
FIG. 7: and (3) qPCR detection results of osteogenic differentiation of stem cells in the high-magnesium microenvironment microspheres. (a) When the stem cells are cultured for 3 days, the expression condition of run-related transcription factor 2 (run-related transcription factor 2, runx 2) of the stem cells in the microspheres is realized; (b) When cultured for 7 days, the microsphere inner stem cell OCN is expressed (spiral:, p < 0.01).
FIG. 8: the experimental result of repairing the critical bone defect of the rat skull by the high-magnesium microenvironment stem cell microsphere carrier is shown in (a) an X-ray and CT three-dimensional reconstruction graph, (b) each group of BV/TV results are analyzed by CT scanning, and (c) each group of BMD results ({ major (b) } 0.01) are analyzed by CT scanning.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, in which specific conditions are not specified, are generally conducted under conventional conditions or under conditions recommended by the manufacturers.
Example 1 detection of toxicity of magnesium ions on rat bone marrow Stem cells
Step one, isolated culture of rat bone marrow stem cells
Sprague Dawley rats of 3 to 4 weeks old were taken, and after neck amputation and death, the rat carcasses were sterilized by immersion in a 75% alcohol solution for 3 minutes. And (4) after the disinfection, obtaining the mesenchymal stem cells in the ultra-clean bench. The skin was cut at the groin of the rat and the tissue was blunt dissected to expose the tibia and femur. Reducing the fracture of the knee joint by using scissors, taking out the tibia and the femur, removing soft tissues such as muscles on the surface of the bone, and the like, and cutting off epiphyses at two ends of the tibia; and (3) sucking a proper amount of DMEM complete culture solution by using an injector, inserting the needle of the injector into the marrow cavity, washing by using the DMEM culture solution, lifting up and down at the same time, ensuring that the content in the marrow cavity is flushed into a centrifugal tube for use, and washing by using the culture solution until the color of the long bone is white. Centrifuging the collected marrow cavity flushing fluid in a centrifuge at 1500-1800 rpm for 10 min; discarding the supernatant, resuspending the cell precipitate with culture solution, uniformly inoculating into a 10cm culture dish, and culturing in a constant-temperature incubator at 37 ℃; after 3-4 days, the cells were passaged when they reached 80% confluence, after observation and fluid exchange. Passage 2 through passage 4 bone marrow stem cells were used in the experiment.
Step two, magnesium ion IC 50 Measurement of (2)
Taking rat bone marrow mesenchymal stem cells, inoculating the rat bone marrow mesenchymal stem cells into a 96-well plate (n = 6) at the density of 5000 per well, and replacing 100 mu L of DMEM complete culture solution containing magnesium ions with different concentrations when the cells grow full (the concentration of Mg ions is 1.6mM, 2.5mM, 5mM, 10mM, 406080mM. After 24h, keeping out of the light, adding 10. Mu.L of MTT reaction solution into each well, carefully mixing, putting the 96-well plate back into the cell incubator to continue culturing for 4h, and then, the bottom of the well is seen to have purple crystals. Removing supernatant by suction, adding 150 μ L of dimethyl sulfoxide (DMSO) into each well, shaking for 10min to obtain purple crystalsDetecting light absorption at 490nm after dissolution, comparing OD value of experimental group with that of control group, calculating cell survival rate, using GraphPad Prism 5.0 software to draw magnesium ion IC50 curve, the experimental result is shown in figure I (a), when magnesium ion concentration is within 20mM, cell activity has no obvious influence, when magnesium ion concentration exceeds 20mM, cell activity is obviously reduced, IC is obviously reduced 50 The concentration was 65.91mM.
Example 2 Effect of magnesium ions on cell bioactivity
Step one, influence of magnesium ions with different concentrations on cell proliferation
DMEM complete culture solutions with magnesium ion concentrations of 1.6, 2.5, 5, 10, and 20mM were prepared according to the magnesium ion concentrations selected in example 1, rat bone marrow stem cells were seeded in a 96-well plate at a density of 1000 cells/well (n = 6), and after the cells were attached to the wall, 100 μ L of high magnesium culture solution was replaced, and the normal DMEM complete culture solution was used as a control group, which was recorded as day 0. In 1, 4, 7 days, 10 mu l of MTT liquid is added into each hole, original liquid in the plate is sucked after incubation for 4 hours, 150 mu l of DMSO is added into each hole, and the vibration is carried out for 10min, so that crystals are fully dissolved. And detecting the absorbance value by using an enzyme-labeling instrument, and analyzing the cell proliferation condition.
The experimental results are shown in fig. one (b), and the proliferation activity of the cells in the high magnesium ion concentration group is not significantly different from that in the control group at days 1 and 4, and the proliferation activity of the cells in each high magnesium ion concentration group is stronger than that in the control group by day 7 (fig. one (b)). The above results indicate that the proliferation of mesenchymal stem cells is facilitated in an environment of high magnesium ion concentration.
Step two, alkaline phosphatase (ALP) staining and alkaline phosphatase semi-quantitative detection
Taking the cells at 5X 10 4 The cells were seeded in 24-well plates at a density of one/mL, and DMEM complete medium containing magnesium ions at different concentrations was replaced after the cells were confluent. After 7 days, the culture broth was aspirated and washed with PBS 3 times, fixed with 4% paraformaldehyde, washed with PBS 3 times, and stained with a staining solution prepared with BCIP/NBT alkaline phosphatase kit. Adding ALP activity semi-quantitative lysis solution into a pore plate, incubating at 37 ℃ until cell lysis is complete, mixing a sample and p-NPP working solution according to a proportion of 1:1, incubating at 37 ℃, and detecting absorbance at 405 nm. Separately, each was measured by BCA methodProtein concentration of the sample, protein mass was calculated from the volume. ALP semi-quantitative activity = OD 405 (ii)/protein mass.
The results are shown in FIGS. two (a), (b), and it was found by ALP staining that the concentration of magnesium ion was 2.5mM-10mM, and the number of ALP staining positive cells was increased as compared with the control group, with 2.5mM and 5mM being most significant. ALP activity semi-quantitative determination found that the ALP activity of the 2.5mM group and the 5mM group was significantly different from that of the control group, and the effect of 5mM was the best. This shows that magnesium ion concentration of 2.5mM-10mM has a certain osteogenic differentiation effect on the stem cells, wherein magnesium ion concentration of 2.5mM-5mM has the best osteogenic differentiation effect on the rat bone marrow mesenchymal stem cells.
Step three, westernblot detection of expression condition of stem cell osteogenesis related protein under magnesium ion stimulation
Taking cells at 1 × 10 6 The cells were seeded at a density of one/mL in a 10cm dish, and after cell fusion, high magnesium medium was added at 2.5mM and 5mM, depending on the semiquantitative results for ALP. After 7 days, protein samples were collected, the supernatant protein concentration was measured by the BCA method, and the sample proteins were adjusted so that the amounts were consistent. Performing SDS-PAGE electrophoresis, transferring the membrane to a PVDF membrane, and sealing with 5% skimmed milk for 1h. OCN antibody was diluted to 1. PBST was washed 3 times, and after incubation of secondary antibody (dilution ratio) at room temperature for 1h, PBST was washed 3 times and developed.
The results are shown in FIG. two (c), and compared with the normal culture solution, the OCN expression levels in the 2.5mM and 5mM groups are obviously improved, wherein the improvement in the 5mM group is more obvious and is consistent with ALP staining and semi-quantitative results.
Step four, immunofluorescence staining detection
Taking cells at 5X 10 5 The cells are inoculated in a glass bottom dish at a density of one cell per mL, and the cells are replaced by DMEM complete culture solution with magnesium ions of different concentrations after being attached to the wall. After 5 days of culture, the old cell culture medium in the glass-bottom dish was blotted dry and rinsed 3 times with PBS buffer. After 4% paraformaldehyde fixation, immunofluorescence staining was performed to detect the expression of cellular OCN. OCN antibody dilution ratio is 1; the dilution ratio of the secondary antibody is 1: :200. After dyeing, the cloth is put in a rollerAfter 30min of staining with rhodamine-labeled phalloidin (dilution ratio 1. The photographs were observed and taken under a fluorescent microscope.
The results are shown in FIG. two (d), and the OCN expression of the stem cells in the 2.5mM and 5mM groups is higher than that of the control group and even higher than that in the 5mM group. The above results all indicate that the high magnesium environment has the effect of promoting the osteogenic differentiation of stem cells.
Example 3 detection of changes in expression of magnesium ion channel MagT1 in Stem cells induced by magnesium ions at different concentrations
Step one, detecting the expression change of stem cell MagT1 after osteogenesis induction
Taking rat bone marrow stem cells with the fusion degree of more than 90%, digesting, centrifuging, counting, inoculating into a 6-well plate and a confocal dish, replacing osteogenic induction liquid (provided by Setarian organisms) when the cell fusion degree reaches 80%, and simultaneously performing osteogenic induction by taking a normal DMEM culture solution as a control (n = 3). Collecting samples after inducing for 3 days and 5 days, and carrying out qPCR detection and immunofluorescence detection, wherein the immunofluorescence staining method is the same as the above method; and adding 1mL of trizol lysed cells into the qPCR sample, collecting the total RNA of each group of samples, measuring the concentration and purity of the extracted RNA under the conditions of 260nm and 280nm of an ultraviolet spectrophotometer, and synthesizing the cDNA according to the instruction of a reverse transcription kit. PCR was then performed to detect the expression of MagT 1. Housekeeping gene GAPDH was used as an internal control. CT values represent Real-time PCR results according to the formula: 2-delta-Delta CT to calculate the relative expression of the gene.
The experimental results are shown in figure three. As shown in fig. three (a), magT1 expression was significantly increased after 5 days of osteogenic induction; fig. three (b) shows that stem cells were osteoinductive for 5 days with elevated OCN and MagT1 expression. This suggests that MagT1 is involved in osteogenic differentiation of stem cells and can be used as an indirect assessment indicator of osteogenic differentiation of stem cells.
Step two, the expression condition of stem cell MagT1 after the induction of magnesium ions with different concentrations
The preparation concentration is 1.6mM;2.5mM;5mM;10mM;20mM;40mM;60mM high magnesium medium, and normal DMEM complete medium as control. Taking rat bone marrow stem cells with good growth condition, digesting, centrifuging and inoculating the rat bone marrow stem cells in a culture dish; after the cells were confluent, the high magnesium medium (n = 3) was replaced, and samples were collected after 5 days for qPCR detection and immunofluorescence staining.
As shown in FIG. three, in FIG. three (c), it was found that the expression of dry cell MagT1 was significantly increased at magnesium ion concentrations of 2.5mM and 5mM, and in FIG. three (d), the immunofluorescent staining result was consistent with the qPCR result, indicating that the magnesium ion-induced bone differentiation was most effective at concentrations of 2.5mM to 5mM.
Example 4 preparation of microsphere vectors for Stem cells of different sizes and evaluation of cell survival
Step one, preparation of stem cell microsphere carriers with different sizes
Taking rat bone marrow stem cells with fusion degree of more than 90%, digesting, centrifuging, re-suspending and counting the culture solution, uniformly mixing with high-temperature and high-pressure sterilized 4% sodium alginate solution in proportion of 1:1-3:1, adjusting cell density to 5 × 10 6 —10 7 After/ml, aspirate with a microsyringe with 0.1M CaCl 2 Is a cross-linking agent, the inner diameter of the syringe needle is 150 μm, the needle and CaCl are controlled 2 The liquid level distance is within 1mm, and the hydrogel-cell mixed solution is dripped into CaCl 2 And (4) carrying out medium crosslinking for 3min, and controlling the volume of the liquid drop within 0.4 mu L. Abandon CaCl 2 After washing with saline for 3 times, DMEM complete culture medium was added, and the microspheres were observed under a microscope to have a diameter of about 500 μm (see FIG. four). Large-size microspheres were prepared with a needle having an inner diameter of about 500 μm, and the microspheres were observed under a microscope to have a size of 1-2mm (see FIG. four). The prepared stem cell microsphere carrier is placed in a 24-pore plate and cultured in a constant-temperature incubator at 37 ℃.
Step two, detecting the survival condition of the stem cells in the microsphere carrier by live-dead staining
And (4) after culturing the microspheres with different sizes prepared in the step one for 3 days, staining the microspheres by using a Calcein-AM/PI live-dead staining kit, detecting the microspheres by using a confocal microscope, and analyzing the survival condition of cells after reconstruction.
The size of the microsphere is related to the inner diameter of a needle head (the composite size can be formed in an experimental selected range, if the microsphere is too large, the microsphere is large, if the microsphere is too small, the extrusion difficulty is large, cells are difficult to survive after being pressed), the distance between a liquid drop and the liquid surface (related to the shape of the microsphere, the shape of the microsphere is oval instead of round when the distance is higher), and the volume of the liquid drop.
The experimental result is shown in the figure IV, after live and dead staining, live cells emit green fluorescence under a microscope, dead cells show red fluorescence, and the figure shows that after 3 days of culture, the dead cells gradually increase along with the increase of the diameter, the cell death rate in microspheres with the diameter of 2mm is close to 50 percent, the fresh cells in the microspheres with the diameter of 500 mu m die, the cell survival rate is obviously improved compared with that in a large-size group, the indication is that the cell survival rate is 500 mu m or less, and theoretically 100 mu m-500 mu m is the optimal size of the microsphere carrier under the current experimental conditions.
Example 5 preparation of microsphere vectors for high-magnesium Stem cells at various concentrations and evaluation of cell survival
Step one, preparation of high-magnesium stem cell microsphere carriers with different concentrations
According to the results of example 2, selecting magnesium ion concentration of 2.5mM and 5mM as experimental groups, taking rat bone marrow stem cells with fusion degree of more than 90%, digesting, centrifuging, resuspending and counting with high magnesium culture solution with different concentrations (normal DMEM control), mixing with high temperature and high pressure sterilized 4% sodium alginate solution at 1:1-3:1 ratio, and adjusting cell density at 5 × 10 6 -10 7 Per ml, final magnesium ion concentrations were 2.5mM and 5mM. Several microspheres of about 500 μm were prepared (same method as example 4), and cultured in normal DMEM medium at 37 deg.C.
Step two, evaluating survival of cells in microspheres
And (3) taking different high-magnesium microenvironment microsphere carriers cultured for 1 day and 3 days, performing living and dying staining, and developing by a confocal microscope. The results are shown in figure five (a), the high magnesium group had less apoptosis at 1 day of culture, and the apoptosis was slightly increased at 3 days, but no significant difference from the control group.
Taking 50 mu L (n = 3) of microspheres of each group cultured for 0 day, 1 day and 3 days, adding the microspheres into a physiological saline solution of 55mM sodium citrate, carrying out shake reaction at 37 ℃ for 5-10 min, centrifuging at 3000rpm for 5min after the sodium alginate microspheres are completely degraded, discarding the supernatant, then resuspending the culture solution, and inoculating the suspension into a 96-well plate. After the cells adhere to the wall, 100 mu L of culture solution is changed, 10 mu L of CCK8 working solution is added into each hole, and the cells are incubated for 1.5h at 37 ℃ in a dark place. After the supernatant is absorbed, an enzyme-labeling instrument detects the OD value at 450nm, and GraphPad Prism 5.0 software analyzes and calculates the cell survival rate. The experimental result is shown in figure five (b), the cell survival rate of the high magnesium group is not obviously different from that of the control group, the survival rate is slightly reduced at 3 days, but still is about 90 percent.
Example 6 Effect of high magnesium microenvironment on Stem cell bioactivity in microsphere Supports
Step one, preparation of stem cell microsphere carrier with high magnesium microenvironment
According to the results of examples 2 and 5, the final concentration of magnesium ions was 5mM. The microspheres were prepared as described in example 4.
Step two, detecting the proliferation condition of stem cells in the microsphere carrier
Preparing an EdU solution with the concentration of 5mg/mL, diluting the solution at a ratio of 1. DAPI stained nuclei for 5min, observed by fluorescence microscopy, edU positive (pink cells) as proliferating cells. Image J software statistical analysis.
The experimental results are shown in fig. six, as shown in (a), when the microspheres are cultured for 1 day, the cell proliferation in the microspheres is less, the cell proliferation proportion is gradually increased along with the increase of time, the cell proliferation proportion is obviously increased on the 7 th day, and simultaneously, the cell proliferation of the 5mM group is more active than that of the control group, which indicates that the high-magnesium microenvironment can promote the proliferation of the stem cells in the microspheres.
Step three, detecting osteogenic differentiation condition of stem cells in microspheres
Taking 50uL (n = 3) of stem cell microspheres cultured for 3 days and 7 days respectively, centrifuging to remove supernatant after dissolving the sodium citrate solution, adding trizol 1mL of lysed cells, collecting total RNA of each group of samples, and detecting the expression of related genes. And measuring the concentration and purity of the extracted RNA by an ultraviolet spectrophotometer at 260nm and 280nm, and synthesizing cDNA according to the specification of the reverse transcription kit. Then PCR is carried out to detect the expression of osteogenic related genes Runx2 and OCN. Housekeeping gene GAPDH was used as an internal control. CT values represent Real-time PCR results according to the formula: 2-delta-Delta CT to calculate the relative expression of the gene.
The results showed that at 3 days, 5mM Runx2 expression was significantly higher than that in control group (Tokyo (a); p < 0.01), and at 7 days, OCN expression was significantly higher than that in control group (Tokyo (b); p < 0.01). The induction effect of the high-magnesium microenvironment on the osteogenic differentiation of stem cells is suggested.
Example 7 Effect of high-magnesium microenvironment stem cell microspheres in repairing Critical bone defect of rat skull
Step one, preparation of stem cell microsphere carrier with high magnesium microenvironment
According to the results of examples 2, 5 and 6, the final concentration of magnesium ions is 5mM; according to the results of example 4, stem cell microspheres with a size of 500 μm were prepared according to example 4 and designated as SA-Mg/BMSCs group; suspending rat bone marrow stem cells by using a common DMEM culture solution to prepare microspheres with the same size, and marking as an SA-BMSCs group; and selecting the final concentration of magnesium ions to be 5mM, preparing the sodium alginate microspheres, and marking as an SA-Mg group, wherein the sodium alginate gel microspheres are a blank control group and are marked as an SA group.
Step two, preparation of critical bone defect of rat skull
Sprague Dawley rats of 6-8 weeks of age were weighed and anesthetized by injection of 10% chloral hydrate solution at a dose of 4. Mu.L/g body weight. After leg-clamping reflection and corneal reflection of a rat disappear, shaving hair, disinfecting a skull top operation area by 75% alcohol, making a sagittal incision along a skull median suture to periosteum, separating the periosteum by a periosteum stripper, and preparing 5 mm-diameter circular bone defects along two sides of the skull median suture.
Step three, implanting the stem cell microsphere carrier
Stopping bleeding with gauze, sucking high magnesium stem cell microsphere carrier (SA-Mg/BMSCs group) with Pasteur tube, dripping to defect area, spreading microsphere uniformly with forceps, drawing periosteum, assisting microsphere retention, suturing periosteum skin in sequence, disinfecting operation area again, observing rat respiration and state, confirming rat state stability, marking, and returning to mouse cage. The same procedure was used for the SA-BMSCs, SA-Mg and SA groups, with a microsphere volume of 50 μ L for each group, and a sample size of n =6 for each group.
Step four, evaluating the repairing effect of the critical bone defect of the skull of the rat
Four weeks after the operation, the rat was euthanized, and a skull specimen was fixed in formalin fixing solution; after fixation, X-ray imaging detection is carried out (the result is shown in figure eight); and then, detecting the new bone formation condition by using a micro CT (computed tomography), and analyzing the bone formation effect of each group.
The results are shown in fig. eight (a), which shows that the SA group has the worst bone repair effect and a large number of defects, while the SA-Mg group and the SA-BMSCs group have partial new bone formation and have little difference; compared with the other three groups, the new bone regeneration of the SA-Mg/BMSCs group is obviously increased, and the repair effect is most obvious. As shown in fig. eight (b), BV/TV (%), SA-Mg/BMSCs group =32.425 ± 4.337, SA-BMSCs group =18.148 ± 3.911, SA-Mg group =14.005 ± 2.864, SA group =3.492 ± 1.103; as can be seen from fig. eight (c), BMD (mgHA/cc), SA-Mg/BMSCs group =344.690 ± 51.246; group SA-BMSCs =213.863 ± 35.839, group SA-Mg =180.706 ± 25.860, group SA =85.622 ± 22.522; as can be seen, the new bone mass in the SA-Mg/BMSCs group was significantly higher than that in the other groups.
In summary, when the magnesium ion concentration is within 20mM, the cytotoxicity is low, and the osteogenesis inducing effect is achieved, and when the magnesium ion concentration is within 2.5mM-10mM, the cell proliferation promoting effect is achieved; when the concentration of magnesium ions is 2.5mM-5mM, the osteogenic differentiation effect of the induced stem cells is optimal; on the basis, the high-magnesium microenvironment stem cell microsphere carrier is constructed, when the microsphere size is 500 mu m, the cell survival rate is high, the proliferation activity is good, and the high-magnesium microenvironment stem cell microsphere carrier has stronger osteogenic differentiation capacity under the stimulation of local high magnesium. When the stem cell microsphere carrier is used for bone defect repair, the differentiation capacity of stem cells is improved under the stimulation of a local high-magnesium environment, the new bone formation amount is obviously improved compared with that of a control group, and the effect of promoting bone regeneration is achieved.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, without departing from the principle of the present invention, a person skilled in the art may make several improvements and modifications, such as loading magnesium-containing particles for the purpose of slow release of magnesium ions and expanding the application range of labeled cells for binding magnesium ions or applying to other researches, and may also achieve the system for maintaining local high magnesium microenvironment by using other known hydrogels, such as common hydrogels of hyaluronic acid, silk protein, chitosan, gelatin, collagen, cyclodextrin, etc., and also for other tissue regeneration repair or in vitro micro tissue construction and related researches, and these improvements and modifications should also be considered as the protection scope of the present invention.

Claims (4)

1. A high-magnesium microenvironment bone marrow stem cell microsphere carrier for bone regeneration is characterized in that magnesium ions are compounded into the microsphere carrier to form a local high-magnesium environment, the size of the microsphere carrier is 100-500 μm, and the concentration of the magnesium ions contained in the microsphere carrier is 2.5-10 mM; the microsphere carrier loads osteogenesis related stem cells, the osteogenesis related stem cells are bone marrow mesenchymal stem cells, and the density of the bone marrow mesenchymal stem cells is 5 multiplied by 10 6 -10 7 Per ml; the local high magnesium environment promotes osteogenic differentiation of the mesenchymal stem cells; the high-magnesium microenvironment bone marrow stem cell microsphere carrier is dripped to a bone defect area for regeneration and repair of bone defect;
the microsphere carrier contains sodium alginate gel, and the final concentration of the sodium alginate gel is 1-2%.
2. The bone marrow stem cell microsphere carrier with high magnesium microenvironment for bone regeneration of claim 1, wherein the concentration of magnesium ions is 2.5mM to 5mM.
3. A preparation method of a high-magnesium microenvironment bone marrow stem cell microsphere carrier for bone regeneration is characterized by comprising the following steps:
(1) Obtaining and culturing the osteogenesis related stem cells; the osteogenesis related stem cells are bone marrow mesenchymal stem cells;
(2) Digesting and centrifuging the osteogenesis related stem cells obtained in the step (1), then re-suspending the osteogenesis related stem cells by using a high-magnesium culture solution, mixing the cell suspension with sodium alginate gel, and dripping the mixture into a cross-linking agent by using a syringe to obtain cell-loaded microspheres, wherein the diameter of each cell-loaded microsphere is 100-500 micrometers, and the concentration of magnesium ions contained in the cell-loaded microspheres is 2.5-10 mM;
the osteogenesis-related stem cells in the cell-loaded microspheresHas a density of 5X 10 6 -10 7 The inner diameter of a needle head of the syringe is 150-180 mu m, a micro sample injector is used for dripping the cell-gel mixed solution, the volume of the liquid drop is within 0.4 mu L, and the distance between the needle head and the liquid level of the cross-linking agent is within 1mm; after the sodium alginate gel used in the step (2) is gelatinized, the final concentration of the sodium alginate gel is 1-2%;
the cross-linking agent is CaCl 2 ，SrCl 2 Or BaCl 2 One kind of (1).
4. The method for preparing the bone marrow stem cell microsphere carrier with the high magnesium microenvironment according to the claim 3, wherein the magnesium ion concentration in the step (2) is 2.5mM to 5mM.
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